Role of neuron–glia interactions in nervous system development: highlights on radial glia and astrocytes

Role of neuron – glia interactions in nervous system development: highlights on radial glia and astrocytes Fla´via Carvalho Alcantara Gomesp and Stevens Kastrup Rehen Departamento de Anatomia, Instituto de Cieˆncias Biome´dicas, Universidade Federal do Rio de Janeiro, Centro de Cieˆncias da Sau´de, Bloco F, Ilha do Funda˜o-21941-590, Rio de Janeiro, RJ, Brazil. p Correspondence address: Tel.: þ55-21-2562-6460; fax: þ55-21-2561-7973 E-mail: [email protected](F.C.A.G.)

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Introduction Neuron – radial glia interactions 2.1. Radial migration 2.2. Role of neuron– radial glia interactions in cell morphogenesis Neuron – astrocyte interactions 3.1. Role of astrocytes in neuronal morphogenesis: implications for neurogenesis, neuronal death and differentiation 3.2. Neuron –astrocyte interactions as mediators of thyroid hormone actions in NS development 3.3. Role of neurons in astrocyte morphogenesis: implications for astrogliogenesis and astrocyte differentiation Concluding remarks

Abbreviations b-Gal: b-galactosidase; BMPs: bone morphogenetic proteins; BrdU: bromodeoxyuridine; CNS: central nervous system; CNTF: ciliary neurotrophic factor; CP: cortical plate; CR: Cajal – Retzius neurons; EGF: epidermal growth factor; EGL: external granular layer; FGF: fibroblast growth factor; GDNF: glial derived neurotrophic factor; GFAP: glial fibrillary acidic protein; GGF: glial growth factor; GFP: green fluorescent protein; IF: intermediate filaments; IGL: internal granular layer; IZ: intermediate zone; ML: molecular layer; NS: nervous system; PDGF: platelet derived growth factor; RGC: retinal ganglion cells; RT –PCR: reverse transcriptase – polymerase chain reaction; SVZ: subventricular Advances in Molecular and Cell Biology, Vol. 31, pages 97–125 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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zone; T3: thyroid hormone; T3CM: conditioned medium derived from T3-treated astrocytes; TGF-b1: transforming growth factor-beta 1; TNF-b: tumor necrosis factor beta; VZ: ventricular zone. Neuron – glia interactions present great complexity and heterogeneity throughout the CNS. They play a crucial role in nervous system morphogenesis from the early stages of neurogenesis and gliogenesis to later stages of establishment of neural connections. A range of epigenetic signals are involved in determination of neural fate potential, lineage specification and cellular differentiation in the CNS and peripheral nervous system. Some of these signals, represented by diffusible factors and cell contact, are derived from neuron-glia interactions. Over the past decade, a considerable effort has been made to elucidate the mechanisms that underlie such interactions. In this chapter, we will focus on neuron-asytrocyte, neuron-radial glia interactions and their implications for cellular morphogenesis.

1. Introduction In 1846, the German pathologist Rudolf Virchow, described for the first time a connective tissue in brain and spinal cord, known as Nervenkitt (nerve glue, Virchow, 1846). To this cellular component, later called neuroglia, was attributed a merely passive, supportive function in the nervous system (NS). At that time, the neuronal doctrine designed neurons as the main functional element of the central nervous system (CNS). As a consequence, research has focused on elucidating cellular and molecular details of neuron biology, whereas glial cells have been regarded as somewhat less important companions to neurons. Today, however, more than a century after their description by Virchow, increasing evidence has accumulated indicating that neurons and glial cells have an intimate and morpho-functional relationship. The vertebrate CNS is composed of two major classes of glial cells: (1) macroglial cells that include astrocytes, oligodendrocytes and embryonic astrocytic precursors known as radial glial cells, and (2) microglial cells. Interactions between neurons and glial components play an important role in several processes of brain development such as neurogenesis (Lim and Alvarez-Buylla, 1999; Song et al., 2002), neuronal proliferation (Gomes et al., 1999b; Oppenheim et al., 2000) and migration (Hatten, 2002; Nadarajah and Parnavelas, 2002), axonal guidance (Garcia-Abreu et al., 1995; Goodman and Tessier-Lavigne, 1997; Shu and Richards, 2001); myelination (Barres, 1997), synapse formation (Na¨gler et al., 2001; Pfrieger and Barres, 1997; Ullian et al., 2001), glial maturation (Gomes et al., 1999a, 2001a; Noda et al., 2000; de Sampaio e Spohr et al., 2002; Yamada and Watanabe, 2002); and neural signaling (Alvarez-Maubecin et al., 2000; Fro´es et al., 1999; Rouach et al., 2000, 2002). One of the most compelling lines of evidence for the role of neuron– glia interactions in the NS development was the identification of the ‘glial cells missing’ (gcm) gene in Drosophila, which functions as a binary switch that turns on glial fate while inhibiting default neuronal fate (Hosoya et al., 1995; Jones et al., 1995). Its mutation causes presumptive glial cells to differentiate into neurons, whereas its ectopic expression forces virtually all CNS cells to become glial cells. Analysis of

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gcm mutants revealed, in addition to a decreased number of glial cells, a series of defects in several axonal tracts. Such defects were attributed mainly to the loss of glial signals important for axonal growth and neuronal proliferation and differentiation. Although a full elimination of glial cells has not been performed in mammals in vivo yet, the recent observation that inducible ablation of astrocytes is associated with a dramatic degeneration of granular neurons in rodent cerebellum is strongly in favor of the obligatory role of astrocyte –neuron interaction in vertebrate NS development (Cui et al., 2001). In addition, cell ablation models reported that in the absence of Cajal –Retzius (CR) neurons, the development of radial glia from cerebral cortex is severely impaired (Super et al., 2000; Xie et al., 2002). Over the past decade, a considerable effort has been made to elucidate the mechanisms that underlie neuron –glia interactions. Although interactions between neurons and oligodendrocytes and/or microglia contribute to NS morphogenesis, it will not be the scope of the present chapter to discuss their implications. In this chapter, we will focus on advances in the understanding of neuron – astrocytic cell interactions during NS development. Firstly, we discuss neuron – radial glia interactions and their implications in neuronal migration and cell differentiation. Briefly, we present some advances concerning the emerging scenario of radial glia and astrocytes as putative neural stem cells. Secondly, we summarize progress in characterizing neuron – astrocyte interactions and their role in several steps of brain development including neurogenesis, neuronal death, differentiation and astrocytic differentiation. 2. Neuron – radial glia interactions 2.1. Radial migration Introduction: radial glia and neuronal migration After exiting the cell cycle, neural cells travel from the germinal zones of the developing CNS to their final positions in the brain. This journey can occur in two different ways: by tangential and radial migration. Tangential migration is termed neuronophilic and is not confined to regional cerebral cortical boundaries. Radial migration, in contrast, is mainly gliophilic and depends on the interactions between migrating neurons and radial glial cells. This latter form of neuronal migration is the most prominent, being characterized by a radial-glia-oriented movement of early-born neurons from the ventricular zone (VZ) to the upper layers of the developing cerebral cortex (see Nadarajah and Parnavelas, 2002, for review). The cells that direct radial migration, radial glia, were first described by Magini, Ramon y Cajal, and others. Interestingly, Magini not only characterized radial glia but also described the occurrence of developing nerve cells along the radial filaments (Bentivoglio and Mazzarello, 1999). However, it was Rakic who first introduced the term ‘radial glia’ in a report examining the fetal primate neocortex using Golgi impregnation and electron microscopy (Rakic, 1972). This study strongly suggested that radial fibers provide a transient scaffold in the developing cerebral wall that promotes neuronal migration.

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Radial glia, as neuroepithelial cells, are located near the ventricular surface and have a characteristic bipolar shape and a long process that guides the migration of young neurons during development (Nadarajah and Parnavelas, 2002). Like astrocytes, these cells express the cytoskeleton proteins, vimentin and RC2 (Misson et al., 1988) and, in the primate cortex, they also express the glial fibrillary acidic protein (GFAP) (Levitt and Rakic, 1980). Nonetheless, the glial nature of radial fibers was controversial (Morest, 1970) until the advent of immunocytochemistry, which confirmed that radial cells are in fact glia (Levitt and Rakic, 1980; Levitt et al., 1981). Actually, the prevailing view of the identity of radial glia is once again under revision as recent studies from several laboratories seem to demonstrate that radial glia are neural stem cells.

Evidence of radial glia as neuronal precursors The development of neuronal and glial cells in the mammalian cerebral cortex has been the subject of investigation for over 100 years. His was the first to recognize the significance of cell migration in development of the NS and, in 1887, to describe the divergence of progenitor cells into the neuronal and glial lineages. Germinal cells visible as mitotic figures lining the lumen of the neural tube would give rise to neurons, while the syncytium of spongioblasts would produce glial cells (His, 1887 in Jacobson, 1991). The traditional view of radial glial cells was as specialized non-neuronal cells, which served solely as migratory scaffolding and disappeared early in development by transforming into astrocytes (Alvarez-Buylla et al., 2002; Parnavelas and Nadarajah, 2001). Recent experimental evidence from several laboratories suggests a surprising new role for radial glial cells as stem cells (Fig. 1; see Gotz et al., 2002 for review). Radial glia express nestin, an intermediate filament protein specifically expressed in neuroepithelial stem cells (Lendahl et al., 1990). They also divide rapidly and undergo interkinetic nuclear migration (Misson et al., 1988). Alvarez-Buylla et al. (1990) first suggested that mitotically active radial glia may give rise to neurons, but the idea failed to gain credence over the prevailing assumption that radial glia were the exclusive precursors of glial cells (Levitt et al., 1981; Alvarez-Buylla et al., 2002 for review). This assumption, however, is now coming under revision, as recent reports have demonstrated that radial glial cells can generate neurons (Hartfuss et al., 2001; Malatesta et al., 2000; Miyata et al., 2001; Noctor et al., 2002). Noctor and colleagues, for example, have investigated whether radial glia are neuronal precursors in vivo. After injecting a green fluorescent protein (GFP)-transducing retrovirus into the lateral ventricules of embryonic rats, the authors discovered clones that included mitotic radial glia and postmitotic neurons arrayed along the glial fiber (Noctor et al., 2001). In addition, radial glia have been shown to generate both neurons and glia in vitro (Malatesta et al., 2000). Radial glia are not the only cells with glial phenotypes that have recently been shown to possess neurogenic potential. Studies of the adult subventricular zone (SVZ) and hippocampal dentate gyrus indicate that astrocytic cells in these regions are neuronal precursors (Campbell and Gotz, 2002; Doetsch et al., 1999; Seri et al., 2001). Further, astrocyte monolayers derived from several regions in the perinatal mouse brain have been

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Fig. 1. Neuronal precursor cells express a mitotic radial glial marker in the VZ of the developing cerebral cortex. Cross-section of a 14-day embryonic mouse cerebral cortex demonstrates that the majority of the mitotic cells identified by the pattern of phosphorylated histone H3 labeling (A) express phospho-vimentin immunostaining (B). Overlay of the two channels (C) indicates that dividing cells in the brain are radial glia. VZ: ventricular zone, IZ: intermediate zone, CP: cortical plate. From M. Kingsbury, S.K. Rehen and J. Chun, unpublished results.

found to generate neurospheres in vitro, which can subsequently differentiate into neurons and astrocytes (Laywell et al., 2000). Thus, the prevailing view of radial glia and astrocytes is in flux: they are no longer seen as merely supporting cells, but rather as cells that give birth to new neurons at the same time as they direct their migration and support their function, respectively (Fig. 2).

Mechanisms and regulation of radial migration Neuronal translocation along radial glial fibers is an interactive, three-step process: first, leading edge extension, then nuclear translocation or nucleokinesis, and finally retraction of the trailing process. Changes in radial glial cell surface properties are thought to signal neurons to cease migration and begin their differentiation at the appropriate location in the developing cortical plate (Anton et al., 1996, 1997). A number of molecules are implicated in the control of young neuron migration along radial glial processes: astrotactin is an adhesion molecule that provides a ligand for neuronal binding to the apposed radial glial fiber. Targeted deletion of astrotactin in mice leads to a slowing of migration in the cerebellum and other defects, including dendritic abnormalities in Purkinje cells (Adams et al., 2002; Zheng et al., 1996). Neuregulins comprise a diverse group of membrane-attached and secreted peptide growth factors that are of central importance to NS development and function (Lemke, 1996). The neuregulins bind to tyrosine kinase receptors ErbB2, ErbB3 and ErbB4 and play a role in neuron – glial interactions during migration. Glial growth factor (GGF), the first isoform of neuregulin shown to participate in neuron – glia interactions, is expressed

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Fig. 2. Radial glial (1) cells are responsible for the production of both newborn neurons (2) and astrocytes (3) that can ultimately become differentiated neurons (4). Radial glia is also responsible for the guidance of its daughter neurons (curved arrow) to their destinations in the developing cortex and as astrocytes, to control neural cell survival (T) and proliferation (dark arrow) in the adult brain.

by migrating cortical neurons and promotes their migration along radial glial fibers (Anton et al., 1997). The current model of neuronal translocation along glial fibers provides the theoretical framework for a series of studies on human cortical neuronal migration malformations, likely those caused by disruptions of the cytoskeleton (for review see Hatten, 2002). Two microtubule-associated protein genes, lis1 and doublecortin (Gleeson et al., 1998) are mutated in lissencephaly, a condition characterized by abnormally shallow cortical sulci and thickening of the gray matter (Cardoso et al., 2002; Crome, 1956). Targeted deletions of lis1 in mice result in embryonic lethality but heterozygotes survive and show slowed cell migration (Hirotsune et al., 1998). The laminar patterning of the cerebral cortex is established when neurons finish their migration by signals from the Reelin pathway. Reelin is an extracellular matrix (ECM) protein, which plays a key role in the organization of architectonic patterns, particularly in the cerebral cortex (Tissir et al., 2002). The recent discovery that the cadherin-related neuronal (CNR) gene family encodes a diverse array of long-sought Reelin receptors (Senzaki et al., 1999) suggests a mechanism for the encoding of a broad range of migratory instructions by Reelin. Other known components of the Reelin pathway include two members of the lipoprotein receptor family: apolipoprotein E receptor 2 (ApoER2) and the VLDL receptor (VLDLR) (Trommsdorff et al., 1999). The intracellular adaptor disabled (mDab1), and integrin a3b1 (Dulabon et al., 2000) also appear to participate in Reelin signaling. The reeler mutation in mice leads to cortical inversion (Caviness, 1976), while in humans reelin deficiency causes a unique phenotype of lissencephaly. Though its

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cellular actions are unknown, reelin may regulate cell adhesion between radial glia and migratory neurons (Hoffarth et al., 1995) and act as a detachment signal, but not a stop or guidance cue (Hack et al., 2002). The formation of the final laminar pattern also requires the integrity of the external limiting membrane, defects of which lead to over-migration of neurons and to human type 2 lissencephaly (Gupta et al., 2002; Lambert de Rouvroit and Goffinet, 2001). Recently, a glycolipid, more specifically the 9-O-acetyl GD3 ganglioside was also shown to have a role in the migration of granule cells in the cerebellum (Santiago et al., 2001).

2.2. Role of neuron – radial glia interactions in cell morphogenesis Besides its well-described function on neuronal migration and corticogenesis, neuron– radial glia interaction plays a pivotal role on radial glia and neuronal development. It is a likely speculation that during the migration route, the intimate contact between migrating neurons and radial cells creates a special environment, which influences cell phenotype. The role of radial glia on neuronal morphogenesis has been investigated in the developing retina where radial cells were demonstrated to be instructive for neuritogenesis (Bauch et al., 1998). Ganglion cells of the chicken retina extend axons exclusively into the inner retina, whereas their dendrites grow into the outer retina. Bauch and collaborators demonstrated that ganglion cell polarity is greatly influenced by different radial glia compartments. However, whereas glial somata induced dendrite formation, neurons cultivated onto glial end feet developed mainly axons (Bauch et al., 1998). Although neuron– radial glia interactions are mainly mediated by contact as previously discussed, a few examples of soluble factors have been reported as mediators of these interactions. Hunter and Hatten have shown that the expression of radial glial cell identity in mammalian forebrain is determined by the availability of diffusible inducing signals (Hunter and Hatten, 1995). Although the factor has not been completely characterized, biochemical studies have indicated that it is different from the neural growth regulators already known. These signals act to transform mature astrocytes into a radial glia phenotype, suggesting that transformation from radial glial cell to astrocyte is reversible. These data provide support for the role of neuronal extrinsic signals in determining and maintaining a radial glial identity and suggest that the transformation of radial glia into astrocytes is regulated by the availability of neuronal signals rather than by changes in cell potential (Hunter and Hatten, 1995). Similar data were obtained from Sotelo’s group, who demonstrated that Bergmann glia differentiation was greatly influenced by Purkinje cells (Sotelo et al., 1994). More recently, by using grafting and coculture systems, they demonstrated that transplantation of embryonic CR neurons into adult cerebella induces a transient rejuvenation of host Bergmann glia into a radial-glia phenotype (Soriano et al., 1997). This process was also shown to be mediated by diffusible factors. Elucidative data on this subject have come recently from ablation assays. Degeneration of CR cells in newborn mice dramatically decreased the number of nestin-positive radial glial apical processes and increased the number of GFAP-astrocytes (Super et al., 2000). These

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findings support an essential role for CR cells in regulating the phenotype of radial glia and the radial glia – astrocyte transformation, a key step for neuronal migration. GGF/neuregulin signaling has also been implicated in neuron –radial glia fiber interactions. Rakic’s group has shown that GGF is expressed by migrating cortical neurons and promotes their migration along radial glial fibers (Anton et al., 1997). Concurrently, GGF also promotes the maintenance and elongation of radial glial cells. In the absence of GGF signaling, radial glial development is abnormal. In erbB2deficient embryos, radial glial fibers formed abnormal end feet, which were minimally arborized. The ability of GGF to influence both neuronal migration and radial glial development in a mutually dependent manner suggests that it functions as a soluble mediator between migrating neurons and radial glial cells in the developing cerebral cortex. 3. Neuron – astrocyte interactions 3.1. Role of astrocytes in neuronal morphogenesis: implications for neurogenesis, neuronal death and differentiation Radial glia have been studied predominantly in the developing brain (Alvarez-Buylla et al., 1990), and although they can persist throughout life in most vertebrates, evidence indicates that most radial glia disappear—transforming into astrocytes (Rakic, 1995)—by the end of neuronal migration. The cellular and molecular events that contribute to this transformation are not known, but the morphological change appears to coincide with the loss of vimentin, RC2 and nestin expression and the acquisition of GFAP immunoreactivity (Alvarez-Buylla et al., 2002; Gotz et al., 2002; Voigt, 1989). In the mature mammalian brain, astrocytes constitute almost one half of the total cell number, providing structural, metabolic and trophic support for neurons. Neuron – astrocyte interactions play a pivotal role in several steps of brain development such as neuronal survival, proliferation and differentiation. Astrocytes represent a potent source for most neurotrophic factors involved in these processes such as FGF, TGF and EGF families (Banker, 1980; Connor and Dragunow, 1998; Gomes et al., 2001a). Members of the EGF and FGF families are potent mitogens for multipotential neural progenitors and are profoundly implicated in several aspects of neurogenesis (Cameron et al., 1998; Kane et al., 1996; Kuhn et al., 1997). Members of the TGF-b family such as TGF-b1 itself and glial cell line-derived neurotrophic factor (GDNF) have been reported to have a broad spectrum of action during NS development (for review see Unsicker and Strelau, 2000). Both are known to favor neuronal survival in vitro as well as in vivo (Bruno et al., 1998; Choi-Lundberg et al., 1997; Tomoda et al., 1996; Oppenheim et al., 2000). Remarkably, astrocytes may also play a negative role in the maintenance of neuronal populations within the brain. Oxidative stress mediated by nitric oxide (NO) and its toxic metabolite peroxynitrite has been previously associated with motor neuron degeneration in amyotrophic lateral sclerosis (ALS) (see Cleveland and Rothstein, 2001 for review). Degenerating spinal motor neurons in familial and sporadic ALS are typically surrounded by reactive astrocytes expressing the inducible form of NO synthase

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(iNOS), suggesting that astroglia may contribute to motor neuron degeneration in ALS (Almer et al., 1999). In fact, spinal cord astrocytes respond to extracellular peroxynitrite by adopting a phenotype that is cytotoxic to motor neurons through peroxynitritedependent mechanisms (Cassina et al., 2002). Moreover, in Alzheimer’s disease (AD) the neurodegenerative changes that are elicited by the accumulation of b-amyloid peptides seems not only to damage neurons directly but also to activate astrocytes (and microglia) to produce inflammatory mediators (Perini et al., 2002—see also the chapter by Barger). The mechanisms that regulate the fate specification of neural stem cells are poorly understood (Gage, 2000). However, the role of astrocytes in promoting adult neurogenesis has been studied in both the SVZ (Lim and Alvarez-Buylla, 1999) and the dentate gyrus (Seri et al., 2001). In the SVZ, migratory neuroblasts and putative precursors are in intimate contact with astrocytes (Doetsch et al., 1997). Culturing dissociated postnatal or adult SVZ cells on astrocyte monolayers supports extensive neurogenesis similar to that observed in vivo. In this case, a direct cell –cell contact between astrocytes and SVZ neuronal precursors seems to be necessary for the production of neuroblasts (Lim and Alvarez-Buylla, 1999). In the hippocampus, astrocytes also actively regulate adult neurogenesis either by instructing neuronal fate commitment or by promoting proliferation of adult neural stem cells (Song et al., 2002). Indeed, the effects of astrocytes seem to be regionally specified: hippocampal or cerebral cortical astrocytes retain the potential to promote neurogenesis, but astrocytes from adult spinal cord do not. These results present an intriguing possibility that the capability for adult neurogenesis might, in part, be due to certain signals provided by regionally specified astrocytes in the adult CNS (Song et al., 2002). During CNS development, neurons must extend projections in order to establish their connections. Growing axons navigate toward their targets in response to a variety of guidance signals in their surrounding environment. These cues include diffusible attractive or repellent molecules secreted by the intermediate or final cellular targets of the axons. Glial cells have been exhaustively reported as a source of asymmetric cues during axonal navigation (Goodman and Tessier-Lavigne, 1997). Commissural and decussation formation in the NS, such as the optic chiasm and the floor plate of the NS, is dependent on the interaction of growing axons and resident glia of these regions. Several adhesion and soluble molecules involved in such interactions have already been reported, such as the laminin-related molecule netrin-1, proteoglycans, ephrins and several ECM proteins. Recently, a new class of proteins, the Slit proteins, have emerged as a pivotal component controlling the guidance of axonal growth cones and the directional migration of neuronal precursors (Piper and Little, 2003). Shu and Richards recently demonstrated that the Slit proteins are implicated in glia-mediated cortical axon guidance during the development of the corpus callosum (Shu and Richards, 2001), when cortical axons from one cerebral hemisphere cross the midline to reach their targets in the opposite cortical hemisphere. A population of midline glial cells act as intermediate guideposts for callosal axons, which avoid this glial region. The authors demonstrated that the chemo-repellent activity of glial cells is due to expression of slit-2 by these cells, whereas cortical axons express their receptors (Shu and Richards, 2001).

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In agreement with in vivo studies, several lines of evidence obtained in vitro have implicated astrocytic soluble factors in neuronal morphogenesis. Neuronal polarity, which is crucial for neural circuits, is modulated to a great extent by glial cells. Sympathetic neurons maintained in vitro in the presence of astrocytes extend axons and dendrites, while in the absence of astrocytes they extend solely axons. Such astrocyte-induced dendritic growth has been reported to be mediated by bone morphogenetic proteins, a subclass of the TGF-b superfamily involved in many aspects of neuronal maturation (Lein et al., 2002; Prochiantz, 1995). Additional evidence for the influence of astrocytes on neuronal morphogenesis has been provided by the studies of Garcia-Abreu and colleagues (Garcia-Abreu et al., 1995, 2000), who demonstrated that astrocytes derived from distinct regions of the midbrain can differently modulate neurite extension. Astrocytes derived from the lateral region of the mesencephalon are permissive to neurite outgrowth, whereas those derived from the midline proved to be restrictive to neuritogenesis (Fig. 3; Garcia-Abreu et al., 1995). These astrocyte populations exhibited great heterogeneity in the content of soluble proteoglycans secreted in the medium, which may account for the differences observed in their ability to support neurite outgrowth (Garcia-Abreu et al., 2000; Mendes et al., 2003). Taken together, the data presented above provide evidence that soluble factors released from astrocytes play an important role in several steps of neuronal morphogenesis from the early events of neuronal precursor proliferation until later periods of neuronal differentiation and establishment of neural circuits. A greater understanding of the interaction between neurons and astrocytes may help advance the therapeutic use of glial cells for both the regulation of neural stem cell proliferation and the modulation of neuronal cell death.

Fig. 3. Heterogeneity of neuron–glia interaction patterns in the mesencephalon in vitro. Mesencephalic neurons cultivated onto astrocytes derived from distinct regions of the mesecephalon: (A) lateral glia, and (B) medial glia. Neurons are marked by b-Tubulin III immunostaining. Note that lateral glia are more permissive for neuritogenesis. Arrows (A) and arrowheads (B) shows neurite length (kindly provided by J. Garcia-Abreu).

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3.2. Neuron –astrocyte interactions as mediators of thyroid hormone actions in NS development Thyroid hormone (3,5,30 -triiodothyronine, T3) is essential for development of the vertebrate NS, influencing diverse processes of brain development such as neuronal migration, neurite outgrowth, synapse formation, myelination and glial cell differentiation (Bernal and Nunez, 1995; Forrest et al., 2002; Gomes et al., 2001b; Lima et al., 2001). The finding that thyroid hormone receptors are present in the developing brain suggests that it exerts its effects by regulating the expression of specific genes (THresponsive genes). (For reviews see Gomes et al., 2001b; Koibuchi and Chin, 2000; Ko¨nig and Moura Neto, 2002.) However, the molecular mechanism of hormone action is still controversial (Anderson et al., 1997; Koibuchi et al., 1999; Potter et al., 2001). Evidence has accumulated over the last 10 years indicating that such endocrine regulation of brain development might be the result of T3-dependent modulation of secretion of several growth factors such as NT3 (neurotrophin 3), NGF (nerve growth factor), IGF (insulin-like growth factor), BDNF (brain derived neurotrophic factor) (Beck et al., 1993; Figueiredo et al., 1993) and FGF (fibroblast growth factor) (Trentin et al., 2001). This proposition is highlighted by the fact that injection of NT-3 or BDNF results in some rescue of cerebellar development in hypothyroid animals (Neveu and Arenas, 1996). The fact that astrocytes have been considered for a long time as a potential source of growth factors makes them a candidate for mediators of T3 action on NS. Several studies have shown that thyroid hormone has direct effects on astroglia in vivo such as regulation of astrocyte number (Clos and Legrand, 1973) and the maturation of Bergmann cells in the rat cerebellum (Clos et al., 1980). A great step towards the understanding of T3-glia-mediated action on neural cells has been provided by the work of Moura Neto and collaborators (for review see Ko¨nig and Moura Neto, 2002). They have demonstrated that treatment of cultured astrocytes by thyroid hormone elicits distinct responses, depending on the origin of the cells. Protoplasmic astrocytes derived from cerebral cortex are morphologically transformed into process-bearing cells upon hormone treatment (Trentin et al., 1995, 1998; Lima et al., 1997). On the other hand, thyroid hormone induces cerebellar astrocyte proliferation in vitro, instead of its morphological differentiation (Trentin et al., 1995; Trentin and Moura Neto, 1995; Lima et al., 1997, 1998). Such effects seems to be mediated by the growth factors secreted by the hormone-treated cells, suggesting an indirect autocrine mechanism underlying the T3 mode of action (for review see Gomes et al., 2001b; Ko¨nig and Moura Neto, 2002). In order to gain insights into T3 effects on the CNS we have focused on the ontogenesis of cerebellum, which is one of the most dramatically affected brain structures in hypothyroidism (Nicholson and Altman, 1972). Most of the granular cells of the cerebellum arise from the external granular cell layer (EGL). Postnatally, these cells migrate from the premigratory zone of the EGL to the internal granular layer (IGL) leaving their axons behind to produce the molecular layer (ML). These events are accompanied by a progressive morphological differentiation of Purkinje cells characterized by perisomatic extensions and dendritic trees (Anderson, 2001; Komuro et al., 2001;

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Miale and Sidman, 1961). Although cerebellar histogenesis is well studied, the molecular mechanisms that control proliferation and differentiation of granular cells are still unknown. These processes have been shown to undergo dramatic modulation by thyroid hormone (Anderson, 2001; Cayrou et al., 2002; Nicholson and Altman, 1972). Besides a series of abnormalities found in the cerebellar cortex, hypothyroidism causes a decrease in EGL proliferation rate, increased neuronal death in the IGL, impaired migration of granular cells, and a deficiency in the elaboration of Purkinje cell dendritic trees, spines and synapses (Nicholson and Altman, 1972). We have recently reported that TNF-b and EGF secreted by cultured cerebellar astrocytes in response to T3 treatment can modulate EGL-neuronal proliferation (Gomes et al., 1999b). Culture of 19-day embryonic cerebellar rat neurons on conditioned medium derived from T3-treated astrocytes (T3CM) increased the neuronal population by 60 – 80% (Fig. 4). Bromodeoxyuridine (BrdU) proliferation assays revealed that this increase was mainly due to neuronal precursor proliferation. The proliferation index was three times higher for neuronal cells maintained in T3CM than in control medium and neuronal survival was not affected by EGF suggesting a prior function for TNF-b and EGF in glia-mediated neuronal proliferation (Gomes et al., 1999b; Martinez and Gomes, 2002). The early germinative zone of the mouse EGL (E15-19) lacks T3 receptors, which will be expressed later in development in the premigratory zone of post-mitotic EGL and IGL (Bradley et al., 1992). These observations highlight the importance of a T3action mediator (possibly glial cells) at least in the early events of cerebellum ontogenesis. Besides modulating neuronal precursor proliferation, thyroid hormone (via astrocytes or not) is also involved in ECM deposition and neuronal migration. The ECM helps to regulate cell migration, survival, differentiation and axonal pathfinding in the NS (Reichardt and Tomaselli, 1991). It has been suggested that astrocytes produce most of the ECM components in the CNS including fibronectin and laminin (Liesi et al., 1983, 1986). In addition, it has been demonstrated that thyroxine (T4) regulates the pattern of integrin distribution in astrocytes by modulating the organization of microfilaments (Farwell et al., 1995) and controls the extracellular deposition and organization of laminin on the surface of astrocytes (Farwell and Dubord-Tomasetti, 1999). Moura Neto’s group has shown that T3-induced cerebellar astrocyte proliferation is accompanied by alterations in the GFAP filaments and fibronectin (Trentin and Moura Neto, 1995). More recently, we provided new insights into T3 modulation of laminin and fibronectin expression (Martinez and Gomes, 2002). Using a neuron –astrocyte coculture model, we have investigated the effects of T3-treated astrocytes on cerebellar neuronal differentiation in vitro. Neurons plated onto T3-treated astrocytes presented a 40– 60% increase of total neurite length and an increment in the number of neurites (Fig. 5). Treatment of astrocytes with EGF yielded similar results, suggesting that this growth factor might mediate T3-induced neuritogenesis. EGF and T3 treatment increased fibronectin and laminin expression by astrocytes, suggesting that astrocyte-induced neurite permissiveness elicited by these treatments is mostly due to the modulation of ECM components (Fig. 5).

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Fig. 4. Soluble factors secreted by astrocytes treated by thyroid hormone induce neuronal proliferation. Conditioned medium derived from thyroid hormone-treated astrocytes (T3CM) increases neuronal population (A) and incorporation of the proliferation marker BrdU (B). Cerebellar neurons were cultivated onto T3CM and CCM (control conditioned medium derived from nontreated astrocytes) and immunostained for the neuronal marker cytoskeleton protein b-tubulin III and BrdU (C,D). Note the presence of dividing neurons double-labeled for both markers (arrow) and nondividing neurons (*) (modified from Gomes et al., 1999b).

Our data on neuronal proliferation and axonal growth together provide evidence that EGF secreted by astrocytes in response to T3 performs a dual role in cerebellar ontogenesis (Fig. 5): acting directly on neurons, EGF promotes proliferation of granular cell precursors; and indirectly, EGF increases neuronal morphological differentiation, by modulating the content of two astrocytic ECM proteins, laminin and fibronectin. Thus, our work gives glial cells a novel role as mediators of the endocrine-regulated cerebellar development and describes an additional role for EGF on brain morphogenesis.

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Fig. 5. (A) Astrocytes treated by thyroid hormone or EGF increase cerebellar neuritogenesis by modulating production of ECM proteins. Thyroid hormone induces astrocyte secretion of EGF. Astrocytic EGF modulates laminin and fibronectin secretion thus strongly enhancing astrocyte permissiveness to neurite outgrowth. (B) Cerebellar neurons from embryonic rats cultivated onto control and EGF-treated astrocyte monolayers. Cells were immunostained using a monoclonal antibody to the neuronal marker cytoskeleton protein, b-tubulin III (modified from Martinez and Gomes, 2002). C, D, and E show fibronectin immunostaining. Note that T3 and EGF greatly potentiated ECM production by astrocytes.

3.3. Role of neurons in astrocyte morphogenesis: implications for astrogliogenesis and astrocyte differentiation A central aim in developmental biology is to elucidate the mechanisms that specify the form of particular cell types during animal development. Although the developmental genetic program is decisive, it does not account for the extraordinary cell-type specific patterns present in the CNS. Within this context, neuron – glia interactions are of fundamental importance in determining cell identity. Only very recently, however, outstanding progress has been made to understand how glial cell fate becomes specified, and what the role is of cell –cell interactions in this process. Further, little is known about

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the molecular mechanisms controlling the differentiation of astrocytes, which is hindered by the considerable diversity and heterogeneity of this cell population. While there is compelling evidence of the effects of astrocytes on neurons, the effects of the neuronal environment on glial cells is still far from being well understood. Most of our knowledge concerning neuron– glial interactions is based on the effects of glial cells on neuronal morphogenesis. However, evidence has accumulated in recent years pointing to a mutual influence between these two cell types. A great part of our understanding concerning effects of neurons on gliogenesis was provided by studies of axon –oligodendrocyte and Schwann cell interactions (for a review, see Barres, 1997). At present, it is widely recognized that the survival and proliferation of oligodendrocytes and Schwann cells are highly dependent on neuronal contact and neuronal soluble factors (Barres, 1997). A variety of newly identified soluble signals, including ciliary neurotrophic factor (CNTF), BMPs and neuregulin-1, were shown to direct multipotential stem cells to become glial cells (McKay, 1997; Morrison et al., 1999). In addition, contact-mediated signaling such as the Notch pathway has been found to induce differentiation of glial cells, including Schwann cells, retinal Mu¨ller cells, and radial glial cells in the cerebral cortex (for a review see Wang and Barres, 2000). Similar neuronal soluble and contact factors, however, are only now beginning to be identified as important cues for astrogliogenesis. Notch is an ancient protein used by vertebrate and invertebrate organisms in controlling multiple aspects of development (Artavanis-Takonas et al., 1995, 1999). During mammalian cerebral cortical development, Notch signaling is implicated early in the prevention of precocious neurogenesis and preservation of precursor pools (Artavanis-Takonas et al., 1995, 1999). Recently, by introducing a retroviral vector containing an activated form of Notch1 (NIC) into the mouse forebrain, Gaiano et al. (2000) demonstrated that the NIC-infected cells became radial glial cells. Gaiano data, together with the observation of Notch1 expression in endogenous radial glia, raise the questions as to how Notch1 is normally activated in this cell type. A tentative explanation given by the authors is that newly generated neurons, expressing high levels of a Notch ligand, activate Notch1 in radial glia during migration along the radial processes. This activation would allow glia to respond to environmental cues, such as GGF or others, which might then maintain glial morphology and gene expression. Studies of Tsai and McKay (2000) found that cell contact helps to regulate the fate choice of cortical stem cells strongly favoring astrocyte generation. Although these investigators did not identify the contact-mediated signal, Notch signaling is an obvious candidate for mediating such a process. Although Notch signaling clearly enhances astrocyte differentiation, its mechanism of action is not clear. One possibility is that by inhibiting neurogenesis, Notch preserves progenitor pools so that the remaining cells can respond to other astrogliogenic cues and subsequently differentiate into astrocytes. Alternatively, Notch might instructively trigger an astrogliogenic pathway by directly activating the transcription of astroglia-specific genes. This idea is in concert with the recent finding that the Notch pathway directly activates the GFAP gene (Ge et al., 2002). Since neurogenesis precedes gliogenesis during NS development, this suggests

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that a molecule expressed by neurons or neuronal precursors might modulate astrogliogenesis (for reviews see Luskin, 1998; Temple, 2001). Whereas knowledge of neuronal effects on astrogliogenesis still suffers from lack of conclusive data, effects of neurons on astrocyte differentiation have proved to be an expanding field over the past decade. The first evidence of neuronal influence on astrocyte morphogenesis was provided by the work of Hatten and collaborators in the 1980s. By establishing a model system to study neuron –glia interactions in vitro, this group clearly demonstrated that granular neurons regulate proliferation and differentiation of cerebellar astroglia. In the absence of granule neurons, cerebellar astroglia assume a flat morphology and proliferate rapidly (Hatten, 1985). Addition of neurons to the cultures rapidly arrested glial cell proliferation and induced its morphological differentiation into profiles resembling the cerebellar astroglia seen in vivo. Although the decrease in proliferation and induction of differentiation occurred simultaneously, those processes were mediated by distinct mechanisms. While neuronal membranes mediate inhibition of glial cell division, glial morphological differentiation requires living neurons (Hatten, 1985, 1987). Several proteins found on neuronal membranes were associated with cell contact regulation of astrocyte functions; however, at that time, the question remained unanswered whether neurons were able to secrete a growth factor that could modulate astrocyte development. Today, however, the list of soluble factors secreted by neurons is being extended, as we will discuss soon. The finding that astrocytes exhibit receptors for a series of soluble factors, previously solely attributed to neurons, enhances the possibility that their development could be modulated by neuronal signals. These signals might include soluble growth factors and neurotransmitter substances. In the following section we will discuss some of the emerging literature on the effect of neuronal soluble factors on astrocyte differentiation. Effect of neuronal growth factors on astrocyte differentiation Some of our knowledge about effects of neuron-derived growth factors on astrocyte biology came from studies of the visual system. The vertebrate eye provides an interesting system to study cell – cell communication. During development, cells from several different sources come together in a coordinated fashion to form the final structure of the eye (Karshing et al., 1986). The retina itself is composed of cells of different origins, where cell numbers must presumably be matched to one another by cell –cell interactions. Most of the cells of the neural retina are generated from multipotential neuroepithelial precursors that reside near the outer surface of the retina. In contrast, retinal astrocytes originate from the optic stalk and migrate across the inner surface of the retina. The migrating astrocytes form a glial network that spreads radially in close association with the retinal ganglion cell (RGC) axons (see also the chapter by Stone and Valter). This invasion by astrocytes has been reported to be mediated by secretion of platelet-derived growth factor (PDGF) by RGCs. This factor is expressed and secreted by RGCs, while PDGF receptor alpha (PDGFRa), an isoform of the tyrosine kinase PDGFR, is expressed in retinal astrocytes. By inhibiting PDGF signaling with a neutralizing anti-PDGFRa or a soluble extracellular fragment of PDGFRa, Fruttiger and collaborators impaired the development of the astrocyte

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network (Fruttiger et al., 1996). Apparently, PDGF mediates a paracrine interaction between RGCs and astrocytes during retinal development. RGC axons exert a strong influence not only on migration of retinal astrocytes, but also on their morphology. At the periphery of the cat retina, where RGC axons are sparse, astrocytes adopt a stellate shape in contrast to the strongly elongated form present in RGCrich regions. Gargini et al. (1998) provided evidence that the axon-dependent morphology of the astrocytes is induced by a signal derived from neuronal spikes. Although the nature of the trophic signal from RGC axons has not been identified, a possibility considered by the authors is that it might be a released polypeptide acting through astrocyte receptors. As far as brain development is concerned, following the morphological data obtained by the Hatten group, an increased amount of evidence has been accumulated pointing towards neurons as modulators of astrocyte gene expression and differentiation (Gomes et al., 1999a; Kvamme et al., 1982; Mittaud et al., 2002; Rouach et al., 2000, 2002; Swanson et al., 1997). A way to study astrocyte differentiation is by evaluating levels of proteins which expression patterns vary during development such as the intermediate filament GFAP (Eng et al., 1971), the enzyme glutamine synthetase (Kvamme et al., 1982) and glutamate transporters (Wu¨rdig and Kugler, 1990), among others. In order to gain insight into astrocyte differentiation induced by neuronal cells, we have focused on GFAP expression. GFAP is the major component of the astrocytic intermediate filaments (IF) (Bignami et al., 1972; Eng et al., 1971; Fig. 6). In general, it is widely

Fig. 6. General pattern of GFAP immunostaining of cultured astrocytes. Cultured cortical astrocytes derived from newborn rats presenting distinct morphology: (A) a protoplasmatic, and (B) a process-bearing astrocyte. Note that GFAP filaments extend from the perinuclear region to all over the cytoplasm (A). In (B), the GFAP filaments are reorganized through cytoplasmic processes (F. Gomes, unpublished results).

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accepted that astrocyte maturation is followed by a switch in IF protein expression. Astrocyte precursors of the rodent embryonic CNS usually express vimentin, which is replaced by GFAP during astrocyte maturation (Dahl, 1981; Pixley and De Vellis, 1984). By using transgenic mice bearing 2 kb of the 50 flanking region of the GFAP gene linked to the b-galactosidase (b-Gal) reporter gene, we have demonstrated that cortical neurons can induce the GFAP gene promoter followed by transgenic astrocyte differentiation in vitro. Addition of embryonic neurons from cerebral hemisphere to transgenic astrocyte monolayers increased by 60% b-Gal positive astrocytes (Fig. 7). This event was dependent on the brain origin of the neurons and was followed by an arrest of astrocyte proliferation and induction of glial differentiation. Addition of conditioned medium derived from cortical neurons had a similar effect, suggesting that a soluble factor derived from neurons might be responsible for the induction of the GFAP gene promoter (Gomes et al., 1999a). Recently, we identified TGF-b1 (transforming growth factor b1) as the major mediator of this event (de Sampaio e Spohr et al., 2002). The TGF-b superfamily comprises multifunctional polypeptide members, which perform critical functions in regulating CNS developmental processes such as cell adhesion, migration and proliferation (Abreu et al., 2002; Bo¨ttner et al., 2000; Massague´, 2000; Unsicker and Strelau, 2000). TGF-b1 inhibits astrocyte proliferation, increases GFAP expression in vivo and in vitro, and modulates several ECM proteins and ionic channels (Laping et al., 1994; Perillan et al., 2002; Rich et al., 1999). Despite the widespread

Fig. 7. Neurons induce GFAP gene promoter and astrocyte differentiation by secreting TGF-b1. (A) An astrocyte culture derived from transgenic mice bearing part of the GFAP gene promoter linked to the bgalactosidase (b-Gal) reporter gene. Cells were immunostaining for GFAP (brown cytoplasm) (arrow) and reacted with X-Gal (blue nuclei, arrowhead) (modified from Gomes et al., 1999a). (B) Addition of neurons, CM or TGF-b1 dramatically increased b-Gal astrocyte number whereas addition of anti-TGF-b1 prevented this effect. G: astrocytes cultured alone; N: astrocytes cultured with embryonic neurons; CM: astrocytes cultured with conditioned medium; TGF-b1: astrocytes cultured with TGF-b1; C: in the absence of neutralizing antibodies to TGF-b1; þ aTGF-b1: in the presence of antibody against TGF-b1.

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effects reported for TGF-b1 in CNS injury, where it has been implicated in the organization of the glial scar (Moon and Fawcett, 2001; Zhu et al., 2002; see also the chapter by Kalman), relatively little has been reported on its role in physiological situations. Recently, TGF-b1 secreted by hypothalamic neurons was reported to modulate the oxytocin receptor in cultured rat astrocytes (Mittaud et al., 2002). Our work was pioneering in revealing a physiological function of TGF-b1 on astrocyte development and GFAP expression. We demonstrated that both cell types, neurons and astrocytes, synthesize and secrete this factor. However, addition of neurons to astrocyte monolayers greatly increased TGF-b1 synthesis and secretion by astrocytes. Further, by taking advantage of the cell culture system we investigated the influence of the developmental stage of neurons and astrocytes on this interaction. We demonstrated that younger neurons derived from 14-day-old embryos of wild type mice were more efficient in promoting astrocyte differentiation than those derived from 18-day-old mouse embryos. Similarly, astrocytes also exhibited a timed responsiveness to neuronal influence with embryonic astrocytes being more responsive to neurons than newborn and late postnatal astrocytes. RT –PCR assays identified TGF-b1 transcripts in young but not in old neurons, suggesting that the ability to induce astrocyte differentiation is related to TGF-b1 synthesis and secretion. Our data support the concept that within the context of brain development, neuronal signals might provide a source responsible for astrocyte development and strongly implicates TGF-b1 in this process. The role of neurotransmitters on astrocyte differentiation In addition to the traditional growth factors, recognition of the role of nonconventional trophic factors such as neurotransmitters on astrocyte differentiation has been growing for the last 10 years. This idea is greatly supported by findings that astrocytes exhibit a large variety of neurotransmitter receptor systems previously thought to be unique to neurons (for a review see Nedergaard et al., 2002; see also the chapter by Hansson and Ro¨nnba¨ck). Several studies demonstrated that the inhibitory neurotransmitter GABA secreted by neurons might act as a diffusible factor inducing the differentiation of neighboring astrocytes (Matsutani and Yamamoto, 1997; Mong et al., 2002). As previously observed by Hatten, addition of isolated neurons to monolayers of cultured astrocytes induced a morphological change (Hatten, 1985). Treatment of cocultures with a GABA-receptor antagonist inhibited the neuron-induced astrocyte differentiation whereas treatment with GABA agonists mimicked the neuronal effect. These results might suggest that GABA released by neurons serves as a signal, which triggers morphological changes in astrocytes (Matsutani and Yamamoto, 1997). Glutamate, the major excitatory neurotransmitter of the mammalian CNS, and its receptors and transporters are likely to be part of the complex network of signals that regulates astrocyte development in vivo. Increasing evidence has demonstrated that synaptically released glutamate could have two different actions on neurons: one direct and well known, through activation of postsynaptic receptors and another indirect, heterocellular action, mediated by the activation of glutamate transporters and receptors on glia, as we will discuss in the following paragraphs.

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Although glutamate has a crucial role in several processes of brain development, excessive accumulation of extracellular glutamate leads to neuronal death and has been implicated in various neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer disease and Huntington disease. The maintenance of normal glutamatergic neurotransmission and the prevention of glutamate-induced neurodegenerative disorders depends primarily on the presence of active glutamate transport in glial cells (McLennan, 1976; Rothstein, 1996; see also the chapter by Schousboe and Waagepetesen). Two main subtypes of glutamate transporters have been described in glia, i.e., GLAST, with expression predominating at early stages, and GLT-1 the expression of which progressively increases with maturity. Swanson et al. (1997) have reported that neurons can modulate astrocyte glutamate transporter expression in vitro. In the absence of neurons, cortical astrocytes maintain polygonal shapes and express only the GLAST transporter. When cocultured with a neuronal layer, many of the astrocytes assume a stellate shape and express GLT-1. These findings support the general principle that normal expression of GLT-1 protein by astrocytes requires a neuronal signal, suggesting that neurons can modulate astrocyte differentiation. Although the nature of this neuronal signal remains to be identified, recent reports have clearly demonstrated that GLT-1 and GLAST expression is modulated by neuronal soluble factors rather than by cell contact (Gegelashvili et al., 2000; Perego et al., 2000). These data suggest a regulatory loop tuning between glutamate physiology in the brain and astrocyte differentiation. Another target in the mediation of neuron –astrocyte interaction are the glutamate receptors, especially the metabotropic receptors, mGlu. They form a family of eight subtypes (mGlu1-8) that have been subdivided into three groups: I, II and III. Members representative of all groups are expressed by astrocytes and are apparently involved in mediation of neuronal signaling (Bruno et al., 1997; Besong et al., 2002; Kommers et al., 2002). Elucidative data on mGluII function has come from works by the Nicoletti group. They demonstrated that activation of group II mGlu receptors in astrocytes in vitro is associated with the presence of neuroprotective factors in astrocyte conditioned medium (see also the chapter by Peng). Medium collected from cultured astrocytes after a brief exposure to mGlu3 receptor agonist is highly neuroprotective against NMDA toxicity (Bruno et al., 1997). Neuroprotection was attenuated after treating the astrocytes with the protein synthesis inhibitor cycloheximide suggesting that astrocytes produce and release a proteic neuroprotective factor in response to mGlu3 receptor activation. More recently, these protective factors were identified as TGF-b1 and TGF-b2 (Bruno et al., 1998; D’Onofrio et al., 2001). Studies in Nicoletti’s group on glutamate physiology together with our own investigations provide new insight on TGF-b1 mediated neuron –astrocyte interactions and create a new scenario for neurotransmitter-growth factor actions on NS development. As illustrated in Fig. 8, a transient activation of group II mGlu receptor in astrocytes leads to an increased production and release of TGF-b1, which in turn protects neighboring neurons against excitotoxic death. Additionally, neurons might also potentiate astrocytic TGF-b1 secretion by release of small amounts of TGF-b1, which activate TGF-b

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Fig. 8. TGF-b1-glutamate putative pathway. Neurons release small amounts of TGF-b1 and glutamate, which act on the astrocytic membrane through TGF-b receptors (TGFbR), glutamate receptors (mGlu) and glutamate transporters (GLT-1 and GLAST). A transient activation of group II mGlu receptor in astrocytes leads to an increased production and release of TGF-b1, which in turn protects neighboring neurons against excitotoxic death. Additionally, neurons might also potentiate astrocytic TGF-b1 secretion by release of small amounts of TGF-b1, which activate TGF-b receptor on the astrocytic membrane. In addition to promoting neuronal survival, TGF-b1 induces GFAP gene expression and the astrocytic differentiation program. Such interaction between TGF-b1 and glutamate signaling suggests that astrocyte differentiation and neuronal development are strictly intricate processes.

receptors on astrocyte membrane. In addition to promoting neuronal survival, TGF-b1 induces GFAP gene expression and the astrocytic differentiation program. Such an interaction between TGF-b1 and glutamate signaling may provide new insights into the mechanism of neuronal degeneration. The interplay between these two pathways may suggest that astrocyte differentiation and neuronal development are processes much more intricate than we previously thought.

4. Concluding remarks Taken together, available literature data support the concept that within the context of brain development, neuron –glia interactions are not of a single type, but rather present great complexity and heterogeneity throughout the CNS. Currently, it is widely accepted that neuron– glia interactions play an important role in several steps of NS morphogenesis from the early stages of neurogenesis and gliogenesis to later stages of establishment of neural connections. A dynamic interplay between neurons and glial cells undoubtedly helps to shape developing neural circuits by controlling the survival and morphology of neurons, the growth of their axons, and the number and efficacy of their synapses. Although we have learned much about the physiology of glial cells over the last 10 years, our knowledge of the function and development of glia is still rudimentary. Several growth factors involved in gliogenesis have been identified and this will certainly be crucial for a better understanding of glial cell functions and interactions with neurons. The recent

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finding that glial cells can act as neural stem cells in the adult mammalian brain clearly highlights the new view of glial cells held by neurobiologists (for a review see Taupin and Gage, 2002). Whereas glial cells have been regarded so far as elements of structural and trophic support, today they might represent a key element in neural cell origin. One key issue in developmental neurobiology is to understand how the brain orchestrates the differentiation of various cell types. A range of epigenetic signals are involved in determination of neural fate potential, lineage specification and cellular differentiation in the CNS and peripheral nervous system. Some of these signals are initiated very early in development due to the diffusible factors and cell contact found in the developmental environment. Within this context, it will be very useful in the future to elucidate the mechanisms involved in Neuronal –glia interactions, since these are among the most relevant interactions neural cells will experience during development. Although several molecules involved in such interactions have already been identified, we are still clueless regarding neuronal effects on glial cells particularly those involved in astrocyte development. It would be worth exploring gene expression in neural cells in order to understand how Neuronal –glia interactions might modulate developmental genes during construction of the NS. Until recently there was no way to fully eliminate mammalian glial cells in vivo in order to explore how the brain develops and functions without them, as done in Drosophila after gcm identification. However, as previously discussed, the recent ablation of astrocytes in the cerebellum and CR neurons in cerebral cortex, which severely impaired granular neuron and radial glial cell development, respectively, clearly demonstrated the mutual dependence of neurons and astrocytes in the mammalian brain. We have taken a great step from the passive glia described by Virchow more than a century ago to the ‘astrocytic stem cells’ of today. The close association between neurons and glial cells during NS development suggests that deep inside these interactions might be hidden the secret of NS organization. Acknowledgements We thank Dhruv Kaushal and Cecı´lia Hedin-Pereira for comments on the manuscript and Marcy Kingsbury for generous help. References Abreu, J.G., Ketpura, N.I., Reversade, B., De Robertis, E.M., 2002. Connective tissue growth factor (CTGF) modulates cells signaling by BMP and TGF-b. Nat. Cell Biol. 4, 599–604. Adams, N.C., Tomoda, T., Cooper, M., Dietz, G., Hatten, M.E., 2002. Mice that lack astrotactin have slowed neuronal migration. Development 129, 965–972. Almer, G., Vukosavic, S., Romero, N., Przedborski, S., 1999. Inducible nitric oxide synthase up-regulation in a transgenic mouse model of familial amyotrophic lateral sclerosis. J. Neurochem. 72, 2415–2425. Alvarez-Buylla, A., Theelen, M., Nottebohm, F., 1990. Proliferation “hot spots” in adult avian ventricular zone reveal radial cell division. Neuron 5, 101– 109. Alvarez-Buylla, A., Seri, B., Doetsch, F., 2002. Identification of neural stem cells in the adult vertebrate brain. Brain Res. Bull. 57, 751–758.

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