Neural stem cells and regulation of cell number

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1 Progress in Neurobiology 66 (2002) 1 18 Neural stem cells and regulation of cell number Lukas Sommer a, Mahendra Rao b, a Institute of Cell Biology, Swiss Federal Institute of Technology, ETH-Hoenggerberg HPM E38, CH-8093 Zürich, Switzerland b National Institute of Aging, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA Received 8 June 2001; accepted 14 September 2001 Abstract Normal CNS development involves the sequential differentiation of multipotent stem cells. Alteration of the numbers of stem cells, their self-renewal ability, or their proliferative capacity will have major effects on the appropriate development of the nervous system. In this review, we discuss different mechanisms that regulate neural stem cell differentiation. Proliferation signals and cell cycle regulators may regulate cell kinetics or total number of cell divisions. Loss of trophic support and cytokine receptor activation may differentially contribute to the induction of cell death at specific stages of development. Signaling from differentiated progeny or asymmetric distribution of specific molecules may alter the self-renewal characteristics of stem cells. We conclude that the final decision of a cell to self-renew, differentiate or remain quiescent is dependent on an integration of multiple signaling pathways and at each instant will depend on cell density, metabolic state, ligand availability, type and levels of receptor expression, and downstream cross-talk between distinct signaling pathways Elsevier Science Ltd. All rights reserved. Contents 1. Regulation of stem cell numbers Regulation of proliferation Proliferation signals Cell death Regulation of programmed cell death Differentiation signals Asymmetric versus symmetric divisions Signal integration by stem cells Acknowledgements References Regulation of stem cell numbers The early-formed neural tube consists of proliferating, morphologically homogeneous cells termed neuroepithelial (NEP) stem cells (Kalyani et al., 1997). NEP cells or neural stem cells (NSCs) are initially present in a single layer of pseudostratified epithelium spanning the entire distance from the central canal to the external limiting membrane (Sauer and Chittenden, 1959; Sauer and Walker, 1959). As development proceeds, NSCs are restricted to the proliferating ventricular zone where they are tightly packed and can communicate with each other via gap junctions (Nadarajah et al., 1998; Rozental et al., 1998). Mitotic activity is accompanied by radial nuclear translocation within the Abbreviations: BMP, bone morphogenetic protein; Cdk, cyclin-dependent kinase; EGF, epidermal growth factor; FGF, fibroblast growth factor; HES, mammalian hairy and enhancer of split homolog; bhlh, basic helix-loop-helix; GFAP, glial fibrillary acid protein; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; mter, mouse telomerase RNA gene; NEP, neuroepithelial; NSC, neural stem cell; NT-3, neurotrophin-3; PCD, programmed cell death; PDGF, platelet-derived growth factor; Rb, retinoblastoma tumor suppressor protein; SCF, stem cell factor; Shh, sonic hedgehog; TGF, tumor growth factor; TUNEL, terminal deoxynucleotidyl transferase-mediated dutp-biotin nick end labeling; VZ, ventricular zone; SVZ, subventricular zone Corresponding ; fax: addresses: (L. Sommer), (M. Rao) /02/$ see front matter 2002 Elsevier Science Ltd. All rights reserved. PII: S (01)

2 2 L. Sommer, M. Rao / Progress in Neurobiology 66 (2002) 1 18 proliferating ventricular zone (Seymour and Berry, 1975). Cell cycle time is short at early stages and increases at later stages of development. Adjacent cells appear synchronized with little difference in individual cell cycle times but some variation across the rostrocaudal axis and dorsoventral axis (Reznikov and van der Kooy, 1995). In response to specific signals, stem cells generate differentiated progeny that lose their attachment to the basal lamina and rapidly migrate to the overlying layers of the cortex. The fetal (or the adult stem cell) is thought to generate all differentiated cells in the nervous system via the generation of intermediate precursors that arise from stem cells through asymmetric division and migrate away from the ventricular zone (VZ) (reviewed in Gage, 2000; Morrison et al., 1997; Panicker and Rao, 2000). These precursor cells can still divide, though their self-renewal capability and differentiation potential is limited compared to VZ NSCs. These partially differentiated cells, termed progenitor cells, begin to express differentiation markers while migrating and can be distinguished from stem cells both functionally and by the expression of specific markers (reviewed in Goldman, 1998; Rao, 1999). More restricted progenitors generate differentiated progeny in a spatial-temporally regulated fashion. In general, neuronal precursors and neurons are born first, followed by oligodendrocyte precursors and subsequently by differentiated astrocytes and oligodendrocytes (reviewed in Alvarez-Buylla et al., 2001; Rao, 1999). As development proceeds, the VZ becomes significantly diminished in size and the number of stem cells that can be harvested is significantly reduced. Moreover, additional germinal zones such as the subventricular zone (SVZ), subgranular zone and the external granular cell layer of the cerebellum develop. Cells in these later appearing germinal layers differ from the early stem cells present in the VZ (reviewed in Rao, 1999). In the adult, stem cells still exist though their exact location is in doubt (reviewed in Alvarez-Buylla et al., 2001; Goldman, 1998; Panicker and Rao, 2000). The adult stem cell arises from the fetal stem cell and its characteristics differ from those of the NEP stem cell (Ciccolini and Svendsen, 1998; Kalyani et al., 1997; Tropepe Fig. 1. Possible stem cell fates. The possible fates of a stem cell are summarized. A stem cell may remain quiescent, enter the cell cycle, undergo symmetric or asymmetric divisions, apoptose or mature into an adult type stem cell under the influence of cell intrinsic mechanisms or extrinsic signals. Two additional fates of stem cells include trandifferentiation to a distinct stem cell that contributes to distinct lineages (reviewed in Panicker and Rao, 2000) and transformation to generate an oncogenic phenotype. Indeed, many tumors are thought to arise as a result of malignant transformation of precursor cells.

3 L. Sommer, M. Rao / Progress in Neurobiology 66 (2002) et al., 1999; Roy et al., 2000a,b). In particular, adult stem cells are quiescent and unlike NEP stem cells express glial fibrillary acid protein (GFAP), a marker characteristic of differentiated glia (reviewed in Alvarez-Buylla et al., 2001). The normal fate of the stem cell in development is summarized in Fig. 1. A stem cell may remain quiescent and simply not enter the cell cycle. Alternatively, it may undergo apoptosis and not contribute to further development. Stem cells may undergo symmetric cell divisions to self-renew or undergo terminal differentiation, or they may undergo asymmetric cell divisions to generate differentiated progeny as well as maintain a pool of stem cells. Finally, a stem cell may transit to another kind of stem cell (e.g. adult stem cell) or transdifferentiate to acquire a distinct set of properties. The total number of stem cells present in the nervous system, or any tissue for that matter, represents a dynamic balance between self-renewal, differentiation and death. This dynamic balance is modulated by excitatory and inhibitory signals to meet the demands of the tissue to which the stem cells may supply newly differentiated cells. The recent ability to identify stem cells and to examine their properties in vitro and after transplantation has allowed a detailed examination of the role of molecules that regulate stem cell number. Both cell extrinsic and cell intrinsic molecules have been identified and their stage-specific roles have been characterized. Factors that regulate growth and survival, factors which bias the choice between self-renewal and differentiation, and factors that regulate fate choices have been described, and these will be discussed briefly below. It is important to point out, however, that little is known about the transition from the quiescent to the dividing cell state of stem cells. It is clear that extrinsic controls exist and that the balance between quiescent and dividing precursors is altered in stress and disease and that the total number of quiescent cells likely declines with time. However, how these generalized signals act to specify the behavior of single cells and how the signal is transduced remain to be determined and will not be discussed here. Readers are advised to consult more specialized reviews (see for example Su, 2000). 2. Regulation of proliferation Small changes in cell cycle kinetics can change the total number of cells generated significantly. Based on the total period of neurogenesis and the number of divisions of precursors during this period (based on cell cycle time) it has been estimated that a stem cell undergoes a total of divisions (Takahashi et al., 1994). Increasing or decreasing the number of precursor cell divisions by even one will double or halve neuronal number, a significant change but a level of variation not normally seen. However, the system also appears to have significant plasticity, and ablation of stem cells or differentiated cells at any given instant is compensated by altering cell division to allow nearly normal recovery (see for example, Doetsch et al., 1999a,b; Kirschenbaum et al., 1999). Overall this suggests that stem cells have a significant replicative capacity that is carefully regulated by extrinsic signals to achieve appropriate total cell numbers. 3. Proliferation signals Several different pathways that interact to regulate cell division have been identified. Perhaps the best understood are those triggered by growth factors. Different growth factors act at different stages of stem cell development and differential growth of stem cells may serve to sculpt the developing brain. All stem cells and precursor cells respond to multiple growth factors, but the exact subset of growth factors acting at a specific stage may be unique for a particular stage of stem cell differentiation. Early neural stem cells respond solely to fibroblast growth factor (FGF), and loss of FGF or FGF receptors leads to a significant reduction in stem cell proliferation (Kalyani et al., 1997; Raballo et al., 2000; Vaccarino et al., 1999a,b). Later appearing stem cells require either FGF or epidermal growth factor (EGF) for proliferation (Gritti et al., 1999; Tropepe et al., 1999). More restricted precursors such as glial precursors appear to respond to platelet-derived growth factor (PDGF) and FGF (reviewed in Lee et al., 2000), while neuronal precursors respond to sonic hedgehog (Shh), FGF, and neurotrophin-3 (NT-3) (reviewed in Venkatraman and Luskin, 2000). The effect of growth factors can be modulated by extracellular matrix components that include heparan sulfate proteoglycans (reviewed in Dutton and Bartlett, 2000). Specific extracellular components are expressed at specific stages in development (see for example, Brickman et al., 1998a,b; Ford et al., 1994), and these VZ components can modulate survival, proliferation and differentiation (Ali et al., 1998; Ferri et al., 1996; Ferri and Levitt, 1995; Miao et al., 1996). While a detailed discussion is impossible, it is important to point out that the effect of both FGF and EGF, the two factors recognized as being important for neural stem cell proliferation, are differentially modulated by the components of the extracellular matrix (Ferri et al., 1996; Ford et al., 1994; Whittemore et al., 1999). These growth factors in turn act on precursor cells to modulate the secretion of extracellular matrix components. For example, Drago et al. (1991) showed that FGFs upregulate steady state levels of laminin B1 and B2 chains. Thus, the extracellular matrix plays an important role in regulating total stem cell number by directly affecting proliferation or modulating the effect of other activators of the cell cycle. Cell adhesion molecules are another class of signaling proteins that play important roles in regulating stem cell behaviors. Neural progenitor cells express several different integrins that appear to be differentially involved in the regulation of proliferation and cell migration/differentiation. Integrins are activated by binding to extracellular matrix proteins such as laminin or to integrins on the surface of other cells, resulting in an intracellular signaling pathway

4 4 L. Sommer, M. Rao / Progress in Neurobiology 66 (2002) 1 18 involving PI3 kinase and Akt kinase. Integrin signaling promotes survival of many different cell types including oligodendrocytes and neurons (Brocco and Panzetta, 1999; Frost et al., 1999). Moreover, proliferation requires activation of 5b1 integrins whereas migration requires activation of 6b1 integrins (Jacques et al., 1998). In addition to growth factors, integrins, and extracellular matrix components, neurotransmitters may also regulate stem cell number. The expression of neurotransmitters, their receptors and transporters has been described during early development (Dammerman and Kriegstein, 2000; Haydar et al., 2000; Olivier et al., 2000) well before the onset of synaptic activity, suggesting important early developmental roles. GABA and glutamate have been shown to directly regulate precursor cell proliferation (Haydar et al., 2000; LoTurco et al., 1995). Stem cells respond to neurotransmitter application with changes in intracellular calcium levels (LoTurco et al., 1995; Owens et al., 2000) as well as alteration in DNA synthesis (Haydar et al., 2000; LoTurco et al., 1995) and gap junction formation (Console-Bram et al., 1998). The effect may be stage-dependent, and differential responses to glutamate and GABA application are seen in VZ and SVZ stem cells (Haydar et al., 2000). VZ stem cells respond with an increase in proliferation while proliferation of SVZ progenitors is inhibited. Neurotransmitter application may also inhibit the effect of growth factors and thus indirectly modulate proliferation. Parnevelas and colleagues (Antonopoulos et al., 1997), for example, have shown that activation of GABA A receptors inhibits the proliferative effect of FGF on cortical progenitor cells. The coupling of receptor-mediated signaling to the cell cycle machinery is the subject of intense study and multiple signal cascades have been implicated. Calcium, camp and PKA signaling have all been shown to regulate proliferation. Perhaps the best studied of these is the ras/raf/mek/erk protein kinase pathway. Activation of this pathway in rodents alters proliferation rates by shortening the G1-S transition time rather than altering G2-M transitions as in early Drosophila development (reviewed in Edgar, 1995). The ras/raf/mek/erk pathway is coupled to the cell cycle machinery via interactions with p53, retinoblastoma tumor suppressor protein (Rb) and E2F families of proteins (Fig. 2). The high self-renewing capacity of stem cells may depend on high levels of p107/e2f1/2 and 3 while expression of necdin, p53, and hyperphosphorylated Rb may be indispensable for terminal differentiation (reviewed in Yoshikawa, 2000). Quiescent stem cells may be held in a transient G0 phase and be capable of reentry into the cell cycle. Cells terminally differentiated may be locked into G0 and therefore be incapable of re-entering the cell cycle. Indeed, activation of the cell cycle machinery in such cells will lead to apoptosis (see below). Most dividing cells respond to proliferative signals for a limited time period. Despite continued exposure to proliferative signals, cells will stop responding and differentiate, undergo apoptosis or senescence. This has been termed the Hayflick limit (Hayflick, 1968). This limited proliferative response is true for some precursor cells also. For example, oligodendrocyte precursors will become resistant to the proliferative effect of PDGF and begin to differentiate despite continued exposure. Likewise a slowing of the proliferative rate of stem cells in prolonged culture has been reported (our unpublished observations). This limit on division appears to be species- and cell type-dependent. Multiple mechanisms that could account for these changes have been described (reviewed in Ishikawa, 2000). These include the accumulation of free radicals and oxidative damage, cumulative effect of replication errors and alterations in telomerase levels. Some stem cells however appear capable of virtually infinite expansion. Some stem cells, however, appear to be immune to the Hayflick limit and will continue to replicate far beyond the normal life span of other somatic cells (including precursor/stem cells). This has been characterized best in ES cells but multipotent stem cells have been maintained in continuous culture for 1 or 2 years, suggesting that multipotent cells do not undergo senescence. Intermediate precursors or blast cells, however, have not been maintained for such prolonged periods and it is likely that they will undergo senescence or fail to respond to proliferative signals (see above). One mechanism that may underlie the ability of stem cells to replicate indefinitely is the expression of the DNA repair enzyme complex that includes telomerase, an RNAdependent DNA polymerase that maintains telomere length. Analysis of telomerase enzyme levels or activity has shown that high levels are expressed by embryonic stem cells, and lower levels are expressed in proliferative cells of renewal tissues. In addition, telomerase activity is downregulated in cells that exit the cell cycle via either terminal differentiation or (reversible) quiescence, and overexpression of the catalytic subunit of telomerase allows cells to maintain a normal karyotype and proliferate for substantially prolonged periods beyond the Hayflick limit. Reduced telomerase activity leads to a loss of telomeric repeats (TTAGGG) at each successive division due to incomplete replication of telomeric ends. Depending on telomere length (which varies from species to species), after a certain number of divisions telomeres may be completely lost with subsequent loss of genes at chromosomal ends or abnormal chromosomal replications leading to loss of proliferative potential or cell death. Telomeres are not just simple physical ends but contain telomere specific proteins (TRF1 and 2) that can modulate telomerase activity (Broccoli et al., 1997), and overexpression can modulate telomere length and consequently proliferation (van Steensel and de Lange, 1997). In addition, however, telomerase activity and levels must modulate the cellular response of stem cells to growth factors. The exact pathway remains to be determined, but accumulating evidence suggests that telomerase signaling is coupled to the cell cycle machinery via the p53 and Rb pathways (reviewed in Ishikawa, 2000). Addition of an antisense Rb to near senescent fibroblasts extends the lifespan by about 10 divisions (Hara et al., 1991). Addition of both p53 and Rb

5 L. Sommer, M. Rao / Progress in Neurobiology 66 (2002) Fig. 2. Proliferation signals. Stem and precursor cells undergo symmetric and asymmetric divisions to generate daughter stem cells. This process is regulated by extrinsic signals that modulate the cell cycle machinery to shorten G1-S transitions. Quiescent stem cells are maintained in a transient G0 phase and can enter G1 under appropriate stimuli. Differentiated cells, particularly neurons, enter a permanent G0 phase andany attempts to reenter the cell cycle lead to apoptosis. The transition from a transient to permanent G0 may involve the upregulation of necdin (reviewed in Yoshikawa, 2000). Oligodendrocytes-like neurons likely enter a permanent G0 phase while astrocytes may undergo a transient growth arrest and may be capable of re-entering the cell cycle under appropriate stimuli. antisense RNAs produces an additive effect with an extension of lifespan by about 20 divisions. Likewise, addition of E6 and E7 that inactivate p53 and Rb, respectively, will restart cell growth in senescent human fibroblasts (Shay et al., 1991). The effect of Rb and p53 seems to be cell type specific (Ishikawa, 2000), and the relative importance of these pathways in neural stem cells remains to be determined. It is important to point out that in addition to regulating telomerase levels, stem cells may have additional pathways for regulating proliferation and telomere ends. While most transformed cells have higher telomerase activity, some cells have no detectable activity and extremely short telomeres. Indeed, oncogenic transformation is increased manifold in telomerase knockout mice (Blasco et al., 1997; Hande et al., 1999). Thus, cells must have developed additional methods to bypass senescence (reviewed in Goyns and Lavery, 2000). Characterization of the telomeric structures in one of the survivor mouse telomerase RNA gene (mter) ( / ) cell lines has shown amplification of the same tandem arrays of telomeric and non-telomeric sequences at most of the chromosome ends. This evidence implicates cis/trans amplification as one mechanism for the telomeraseindependent maintenance of telomere ends in mammalian cells. It remains to be seen, however, if telomeraseindependent mechanisms contribute to stem cell proliferation and self-renewal. A final aspect of the regulation of proliferation of stem cells is regulation of the overall size of the tissue or organ relative to the overall size of the organism. Clearly such

6 6 L. Sommer, M. Rao / Progress in Neurobiology 66 (2002) 1 18 global mechanisms exist. However, our understanding of these mechanisms remains limited. They are nevertheless important in considering xeno stem cell transplants and need to be considered when studying the properties of stem cells in different species. Overall the literature suggests that multiple proliferative signals that are cell type- or developmental stage-dependent act on stem cells to regulate their proliferation. The effect of these signals is coupled to the cell cycle machinery via E2F, p53 and Rb pathways, and different members of these families are important in different cell types. The overall effect of proliferative signals is modulated by more global signals such as the oxidative status, the levels of telomerase enzymatic activity, accumulation of replicative errors and diffuse signals that couple growth and proliferation. 4. Cell death Regulation of neuronal cell number and connectivity by programmed cell death (PCD) is a well established and important aspect of normal development at later embryonic stages (reviewed in Deshmukh and Johnson, 1997). At earlier stages of brain development, region-specific cell death has been implicated in morphogenetic processes such as the closure of the hindbrain neural tube and the segment-specific elimination of rhombencephalic neural crest (reviewed in Graham et al., 1996; Kuan et al., 2000). In addition, however, PCD may also be important in regulating stem cell number at the very earliest stages of development (reviewed in de la Rosa and de Pablo, 2000; Gilmore et al., 2000b). Due to the exponential expansion of a single progenitor cell, elimination of stem cells or precursor cells by PCD at early stages of development will have a marked effect on the final number of terminally differentiated cells (Fig. 3). The occurrence of death in proliferative neural cells was predicted from lineage tracing experiments using recombinant retroviruses (Acklin and van der Kooy, 1993) and has been confirmed by labeling of dividing cells of the VZ with terminal deoxynucleotidyl transferase-mediated dutp-biotin nick end labeling (TUNEL) (Thomaidou et al., 1997), a technique that labels DNA fragmentation in apoptotic cells (Gavrieli et al., 1992). However, the estimates of the extent of PCD in early neural development are Fig. 3. Apoptotic signals in stem cells. Programmed cell death (PCD) regulates cell number at several stages during neural development. While the processes leading to apoptosis of differentiated neurons have been well characterized, the signals inducing cell death in stem cells are poorly understood. They likely include withdrawal of survival factors or exposure to death signals. These death stimuli initiate signaling pathways that are cell type-specific and signal-dependent. Some death signaling components, such as Apaf-1, caspase-9, and caspase-3, have been shown to be important for PCD in the proliferative neuroepithelium.

7 L. Sommer, M. Rao / Progress in Neurobiology 66 (2002) divergent (Chun, 2000; Gilmore et al., 2000a). While Chun and coworkers applied a very sensitive method to suggest surprisingly widespread cell death in the proliferative neuroepithelium (Blaschke et al., 1996, 1998), others judge PCD to be less prominent based on microscopic analysis and TUNEL labeling (reviewed in Gilmore et al., 2000b). The magnitude of PCD might be difficult to assess due to the short clearance time of apoptotic cell corpses in the developing brain (Raff, 1998; Thomaidou et al., 1997). While its relative magnitude or its precise role remain unclear, the importance of PCD during early stages of neural development has been inferred from the analysis of mutant mice deficient in components of the cell death signaling pathway. Caspases are central players in activating and executing PCD (reviewed in Deshmukh and Johnson, 1997; Raff, 1998). Among the more than 14 caspases isolated so far, caspase-3 (Pompeiano et al., 2000; Roth et al., 2000) and caspase-9 (Hakem et al., 1998; Kuida et al., 1996, 1998) are expressed in telencephalic cells within the VZ. Activation of caspase-3 leads to progenitor cell death, and blocking caspase-3 activation by a pan-caspase inhibitor prevents PCD, indicating that neural progenitors possess a caspase-dependent apoptotic pathway (D Sa-Eipper and Roth, 2000). The majority of homozygous caspase-3 and caspase-9 null mice die perinatally with a marked hyperplasia and ectopic mass formation in various brain regions including cortex, cerebellum, striatum, hippocampus, and retina. The hyperplasias have been attributed to a reduction in apoptosis in precursor cells with a consequent increase in the number of proliferating cells (Hakem et al., 1998; Kuida et al., 1996, 1998). In addition, mice carrying a mutation in Apaf-1 that encodes another proapoptotic factor of the PCD signal transduction pathway display a very similar phenotype (Cecconi et al., 1998; Yoshida et al., 1998). The timing of death and the selective VZ enlargement suggest that Apaf-1, caspase-9, and caspase-3 functions are essential for apoptosis during early development of the CNS and that in the absence of normal PCD precursor cell development is abnormal. While the antiapoptotic molecule Bcl-X and its inhibitor Bax modulate the PCD signal transduction pathway in postmitotic neurons, these factors do not regulate apoptosis in progenitor cells. Animals with these genes mutated do not show a VZ enlargement or hyperplasia (Motoyama et al., 1995; Roth et al., 2000; White et al., 1998) suggesting that different members of the caspase family are important at different stages in development and that the composition of apoptotic pathways is cell type-specific. In addition to stage-specific differences, there might exist regional differences in cell death pathways since neural progenitors of the neural tube are not (or much less) affected than those of more anterior brain regions in caspase-9 and caspase-3 mutant embryos (Hakem et al., 1998; Kuida et al., 1996, 1998). The cell types affected in the different brain regions remain to be determined. The available data leave open whether stem cells or more restricted proliferating progenitor cells undergo cell death during normal development. A reduced loss of quiescent stem cells, proliferating stem cells or progenitor cells would result in the same apparent phenotypes (Fig. 3). In any case, however, the combined results provide strong evidence for an important role of PCD in regulating the size of the progenitor population in the central nervous system, a function that is distinct from the classic role of cell death in matching postmitotic neuronal populations with postsynaptic targets. 5. Regulation of programmed cell death The occurrence of PCD in the proliferative neuroepithelium raises the question of how the balance between maintenance and depletion of the progenitor pool size is appropriately controlled by the extracellular environment. In postmitotic neurons, cell survival and death are regulated by competition for extrinsically supplied neurotrophic growth factors (reviewed in Deshmukh and Johnson, 1997). Withdrawal of these growth factors induces cell death, but the stimuli that initiate these events in neuronal apoptosis remain to be elucidated. The regulated association of growth factor receptors with antiapoptotic proteins such as BAG-1 might be involved (Bardelli et al., 1996). Recent data suggest that trophic deprivation of motoneurons is accompanied by active triggering of PCD involving signaling by the death receptor Fas (Raoul et al., 2000). In addition to a competitive loss of trophic support, there is growing evidence that the cell number in the developing nervous system is also determined by extrinsic factors that actively promote cell death (Frade and Barde, 1998; Raoul et al., 2000). These include among others oxidative stress, changes in lipid composition, and exposure to neurotransmitters such as glutamate and dopamine. In addition, certain proteins such as LIF, CNTF and TGF promote neuronal apoptosis (Frade and Barde, 1998; Krieglstein et al., 2000; Raoul et al., 2000; Sastry and Rao, 2000). Likewise, activation of p75 by neurotrophic factors mediates PCD of specific neuronal cell types (Bamji et al., 1998; Frade and Barde, 1999; Frade et al., 1996). It is likely that the size of neural progenitor populations is controlled by trophic factor availability as well as by specific death signals. Distinct classes of multipotent, self-renewing stem cells in the CNS are supported in culture by distinct survival factors (reviewed in Rao, 1999). As discussed before, FGF-dependent neuroepithelial stem cells (NEP cells) give rise to various neural cell types and, in addition, they produce another type of CNS stem cell that requires EGF as a survival factor. The survival of NEP cells appears dependent on FGF availability (Kalyani et al., 1997; Mujtaba et al., 1999). Increasing FGF results in increased survival while reducing FGF levels by either removing FGF or the receptor results in a marked reduction in VZ size (Tropepe et al., 1999; Vaccarino et al., 1999a,b; reviewed in Rao, 1999). It remains to be shown, however, whether in

8 8 L. Sommer, M. Rao / Progress in Neurobiology 66 (2002) 1 18 vivo FGF and EGF are survival factors for neural stem cells that counteract naturally occurring PCD in the proliferative neuroepithelium. Such an antiapoptotic function has been demonstrated for insulin in the neuroretina (reviewed in de la Rosa and de Pablo, 2000). Insulin prevents PCD in growth factor deprived proliferating neuroepithelial cells derived from chick retina (Diaz et al., 1999) whereas blocking insulin signaling by in vivo antibody treatment increased PCD in areas of the neuroretina that also undergo naturally occurring cell death (Diaz et al., 2000). It is interesting to note that NEP cells require insulin and IGF for their survival in vitro. Thus, as described above for postmitotic neurons, absence of survival factors might represent one cause of PCD in neural progenitor cells. However, the molecular mechanisms triggering apoptosis in the neuroepithelium are not understood. The activity of c-jun N-terminal kinases (Jnk) possibly couples external stimuli (or their absence) to PCD in early neural development. This assumption is based on the recent finding that caspase-3 activation and cell death are considerably increased in the forebrain neuroepithelium of mice embryos deficient for both Jnk1 and 2 (Kuan et al., 1999). Thereby, the influence of Jnk activity on PCD appears to be context-dependent since in contrast to the forebrain VZ, absence of Jnk1 and 2 results in reduced morphogenetic apoptosis in the hindbrain. Although Fas death receptor appears to play a role in active killing of postmitotic neurons (Raoul et al., 1999), it is unlikely to elicit PCD in early neural progenitor cells. Fas expression during nervous system development is relatively late and not found in the VZ (Cheema et al., 1999). Rather, the release of cytochrome c from mitochondria and the subsequent activation of an Apaf-1/caspase-9 complex might constitute the apoptotic pathway in the proliferative neuroepithelium (Schulze-Osthoff et al., 1998) (Fig. 3), given the abovementioned effect of Apaf-1 and caspase-9 on early neural PCD (Gilmore et al., 2000b; Kuan et al., 2000). To date, only few death signals for multipotent neural stem cells or progenitor cells have been described. Neurotrophins induce death in SCF-treated neural crest cells that express the low-affinity neurotrophin receptor p75 but none of the trk receptors (Sieber-Blum, 1998). Thus, similar to its role in postmitotic neurons (Frade and Barde, 1998; Raoul et al., 2000), p75 might possibly act as death receptor in multipotent neural crest cells, although this has not yet been confirmed in vivo. TGF isoforms can also act as proapoptotic factors on multipotent neural crest-derived progenitor cells provided that the cells receive additional signaling by short-range cell cell interactions (Hagedorn et al., 1999, 2000). The expression of a TGF isoform in differentiating peripheral neurons in vivo (Flanders et al., 1991; Hagedorn et al., 2000) is consistent with the idea that neuron-derived signals regulate the fate and number of surrounding multipotent progenitor cells during peripheral gangliogenesis (Sommer, 2001). 6. Differentiation signals The neuronogenetic interval in mouse spans 6 days (Takahashi et al., 1994). In the course of these 6 days the founder population and its progeny execute 11 cell cycles. With each successive cycle there is an increase in the fraction of postmitotic cells that leaves the cycle (the Q fraction) and also an increase in the length of the cell cycle due to an increase in the length of the G1 phase of the cycle (Cai et al., 1997a; Miyama et al., 1997). Gliogenesis commences a little bit later and continues throughout life (Altman, 1966; Sturrock, 1982). Glial precursors have been first detected at E13.5 in rats (Rao and Mayer-Proschel, 1997; Spassky et al., 1998) followed by astrocytes at E16 and oligodendrocytes a few days later (Timsit et al., 1995; Tohyama et al., 1991). Retroviral labeling and clonal culture data have shown that the same stem cell can generate neurons initially followed by glial progeny later (Levison and Goldman, 1997) and that this process of differentiation involves a sequential series of differentiation steps (reviewed in Rao, 1999). Neurons and oligodendrocytes appear to be terminally differentiated and incapable of re-entering the cell cycle (see Brewer, 1999, however, for an alternate view) while astrocytes appear to be held in a transient G0 phase and capable of re-entering the cell cycle. Differentiation can be thought to involve multiple steps (Fig. 4) that include regionalization, instructive and inhibitory differentiation cues, and factors that regulate exit from the cell cycle. In general, it is thought that precursor cells relatively early in development are specified along the rostrocaudal and dorsoventral axis by homeotic and other patterning signals (Fig. 4). Accumulating evidence suggests that these signals act to alter the properties of the stem cell populations themselves. Thus, at early stages homeobox genes may act to transform the positional identity of stem cells and, as a consequence, the tissue or region that will develop. For example, Hox-1 misexpression in chicken embryos is sufficient to alter rhombomere3 identity (Bell et al., 1999). Likewise, misexpression of Pax-6, eyeless and other eye-specific genes is sufficient to induce ectopic eye formation (reviewed in Gehring, 1996). Transplant experiments have suggested that dorsoventral and rostrocaudal specification occurs at the stem cell stage and that stem cells isolated from different regions consequently differ in their differentiation bias. For example, progenitors from the hippocampus of newborn mice, but not from the cerebellum or midbrain, produce hippocampal pyramidal neurons (Shetty and Turner, 1998). Mesencephalic neural precursors (Ling et al., 1998) differentiate into predominantly dopaminergic neurons while other precursor cell populations do not (reviewed in Svendsen and Rosser, 1995). Medial ganglionic eminence (MGE) precursors differ from lateral ganglionic eminence (LGE) precursors in both their migration and differentiation ability (Wichterle et al., 1999). Thus, as embryonic development proceeds, stem cells themselves may undergo regionalization to acquire distinct properties while

9 L. Sommer, M. Rao / Progress in Neurobiology 66 (2002) Fig. 4. Differentiation signals. Differentiation involves integration of a complex cascade of signals that may act to define the positional identity of the cell, permit a cell to respond to other specific signals (permissive factors) or act to bias differentiation (instructive factors). The effect of these factors is tightly coupled to factors that regulate proliferation. Note we have presented a linear differentiation process for simplicity. It is likely that homeotic regulation, alterations of proliferation rates and response to differentiation signals may be regulated simultaneously and, indeed, on occasion may be regulated by a single factor. maintaining their proliferative potential and ability to differentiate into multiple phenotypes. This view has been challenged, however, by studies in which cerebellar precursors from newborn rats or immortalized NSC lines were able to acquire features characteristic for the host tissue upon transplantation (Vicario-Abejon et al., 1995; Shihabuddin et al., 1995; Flax and Snyder, 2000; Snyder et al., 1992). Maintenance in culture and immortalization prior to transplantation likely affect the plasticity of stem cells. Optimally, the developmental potential of neural stem cells should thus be tested by transplantation of cells prospectively purified from uncultured tissues (Anderson et al., 2001). Later in development a decision is made by regionalized multipotent precursors to generate a particular cell type, such as the progenitor of a neurogenic lineage as opposed to that of a glial lineage (Fig. 4). Among the genes that influence this decision are proneural genes (reviewed in Brunet and Ghysen, 1999). Much of our information on the role of proneural genes in fate decision and early differentiation comes from work performed in Drosophila (reviewed in Chitnis, 1999). Vertebrate homologues of many of these transcription factors often act in cascades to control the successive steps in neuronal determination and differentiation as well as neuronal-subtype identity (Brunet and Ghysen, 1999; Okano et al., 1997). Moreover, the activity of proneural genes inhibits gliogenesis in the CNS (Tomita et al., 2000), while mammalian hairy and enhancer of split homolog (HES) gene expression promotes a glial fate (Furukawa et al., 2000; Hojo et al., 2000; Satow et al., 2001). It is likely that other genes also influence multipotent stem cells to become glial precursor cells (Lee et al., 2000). In Drosophila, glial cell missing (GCM) regulates glial fate decisions, but rodent GCM homologues do not appear to regulate glial differentiation (reviewed in Sommer, 2001). In contrast, the expression of Olig1 and Olig2 suggests a role of these basic helix-loop-helix (bhlh) factors in the early development of oligodendrocytes (Lu et al., 2000a,b; Zhou et al., 2000). A recent study of mice carrying an Nkx2.2-null mutation indicates that this transcription factor is involved in oligodendrocyte differentiation rather than specification (Qi et al., 2001). In the PNS, peripheral glia fail to form in the absence of the transcriptional modulator Sox10 (Britsch et al., 2001). Analysis of the developmental potential of Sox10-mutant neural crest stem cells revealed that Sox10 is not only required for the survival of at least a subset of neural crest cells but also for glial fate acquisition (Paratore et al., 2001). Intermediate neuronal and glial precursors then generate differentiated progeny that exit the cell cycle (Fig. 4). This exit from the cell cycle appears to be both positively and negatively regulated. Wnt, HES, Id and other inhibitory molecules can prevent differentiation without altering cell cycle times, and perturbing the function of these factors can impede expansion of progenitors and promote premature differentiation (Ikeya et al., 1997; Nakamura et al., 2000; Norton, 2000; Ohtsuka et al., 2001; Toma et al., 2000). Other proliferative signals inhibit differentiation as a byproduct of their activation of the cell cycle. Some factors such as bone morphogenetic protein (BMP) and retinoic acid can act as specific inhibitors of proliferation, and these factors regulate the levels of cyclin-dependent kinase (Cdk) inhibitors such as p15, p16, p21 and p27 (see Fig. 2). These inhibitors in turn inhibit proliferation by inhibiting phosphorylation of the Rb protein (reviewed by Weinberg, 1995a,b). The terminal division of many neural cells appears to be a sensitive response to the level of active p27 (Edlund and Jessell, 1999). Mice lacking p27 display disturbed organization of retinal cell layers (Nakayama et al., 1996). In Xenopus, overexpression and antisense experiments indicate a role of p27 not only in inhibiting cell division of neural cells but also in promoting Müller glia from retinoblasts (Ohnuma et al., 1999). Analysis of p27-deficient mice suggests, however, that p27 is not required for cell fate specification or for the onset of differentiation in the mouse retina but rather to regulate the progenitor number (Levine et al., 2000). Absence of p27 activity in oligodendrocyte development reveals a role of p27 in timing the cell cycle exit (Casaccia-Bonnefil et al., 1997, 1999; Durand et al., 1998) and also results in

10 10 L. Sommer, M. Rao / Progress in Neurobiology 66 (2002) 1 18 delayed oligodendrocyte differentiation in vitro (Durand et al., 1998). Similarly, p27 provides a link between cell proliferation and sensory hair cell differentiation and is involved in maintenance of quiescence in non-neuronal cells of the developing inner ear (Chen and Segil, 1999). Prox-1, PNG-1, Hu, some members of the Rb family, and some cyclins are rapidly expressed when cells exit the cell cycle. Several other factors such as nestin, Mushashi-2, BFBP, Sox2, etc. appear to be expressed solely in the proliferating cells and after withdrawal from the cell cycle are rapidly downregulated in a period of hours. Thus, initiation of differentiation involves a complex cascade of events that are carefully orchestrated to generate appropriate differentiated progeny. While simply arresting division by growth factor withdrawal will promote differentiation, the fate differentiated cells adopt appears to be independent of the factor used to inhibit proliferation and depends to some extent on the developmental state of the cell. Thus, BMPs may inhibit proliferation and cells may differentiate into neural crest (Mujtaba et al., 1999), postmitotic neurons (Kalyani et al., 1998) or astrocyes (Mabie et al., 1997) depending on the differentiation state of the precursor cell. Likewise, the effects of retinoic acid are cell type and stage-specific (Cheung et al., 1997; Maden and Holder, 1992; Schulman and Evans, 1997). However, while exit from a proliferative state and acquisition of a particular differentiated phenotype appear to be independently regulated events, exit from the cell cycle needs to be finely coordinated with the process of differentiation. Lack of coordination may lead to apoptosis (see above). Once stem or precursor cells have undergone differentiation to a particular phenotype additional molecules play a role in specifiying further characteristics of a particular differentiated cell type. For example, in a neurogenic lineage, a decision may be made to generate a specific neuron type such as a sensory or motor neuron. POU homeodomain proteins appear to be an important class of regulatory molecules that act at this stage to specify neuronal or glial identity (McEvilly and Rosenfeld, 1999). The LHX, HLH and islet families of proteins appear to be important in specifying specific neuronal sublineages (Lumsden, 1995; Thor et al., 1999). While a detailed discussion of these downstream effectors is outside the scope of this review it is important to note that, in general, multiple genes can affect neuronal fate and these genes can act in different ways to influence the development of different types of neuronal cells (reviewed in Bang and Goulding, 1996; Jessell, 2000). Alterations at these later stages of development have little measurable effect on stem cell number or proliferation rates though subtle changes cannot be ruled out given the technical limitations of such assessments. 7. Asymmetric versus symmetric divisions Multipotent cells in the VZ undergo both symmetrical and asymmetrical divisions. Cai et al. (1997b) used retroviral labeling to show that approximately 48% of labeled cells formed clusters located entirely within the VZ suggesting self-renewal via symmetrical divisions. Approximately 20% of cells, however, appeared to generate cells in both the VZ as well as in the mantle suggesting at least some asymmetrical divisions. Symmetrical divisions that generate two stem-like daughter cells will double the pool of dividing cells, while symmetrical divisions that generate differentiated progeny will remove dividing cells and halve the dividing cell pool. Asymmetrical divisions will maintain the size of the pool while generating differentiated progeny. If a stable precursor pool needs to be maintained then the balance between asymmetrical and symmetrical division must be tightly regulated. Several molecules that may be important in this process have been identified. These include asymmetrically distributed molecules such as Notch and Numb, as well as cytoskeletal components that regulate intracellular movement of molecules, and proteolytic enzymes that regulate asymmetrical distribution by degrading molecules in unwanted compartments. Notch may be an important molecule in specifying cell fate (Baker, 2000; Wang and Barres, 2000). Four mammalian homologues have been identified, and alterations in Notch-1 and Notch-3 affect stem cell fates (Beatus et al., 1999; Furukawa et al., 2000; Gaiano et al., 2000; Lardelli et al., 1996; Morrison et al., 2000; Wakamatsu et al., 1999, 2000). Notch may also be asymmetrically distributed in stem cells (Chenn and McConnell, 1995; Wakamatsu et al., 2000), and cells that have a higher level of Notch continue to divide while those with lower levels differentiate. Numb, a putative Notch antagonist, interacts with the cytoplasmic domain of Notch and might play a role in cell determination (Verdi et al., 1996; Wakamatsu et al., 1999; Zhong et al., 1996). Numb is expressed in stem cells and is asymmetrically distributed (Wakamatsu et al., 1999; Zhong et al., 1996). In addition to its asymmetrical distribution, splice variants may be functionally important. Numb isoforms that contain the long PRR domain promote proliferation while isoforms lacking this domain promote differentiation (Verdi et al., 1999). Targeted deletion in mice demonstrates a role of numb in neurogenesis although the early lethality of the mutant embryos precludes a thorough analysis of the cellular function of numb (Zhong et al., 2000; Zilian et al., 2001). It has been shown that Notch and Numb (and possibly other molecules) are distributed apically or basally and will be differentially partitioned into the two daughter cells depending on the axis of division (reviewed in Lu et al., 2000a,b). Divisions in the horizontal plane will result in asymmetrical localization of Notch and Numb to the two daughter cells with consequent differences in cell fate. Divisions in an oblique or perpendicular plane will result in a more equal partitioning with a greater likelihood of symmetric divisions. How the axis of cell division is determined is a subject of study and possible mechanisms are discussed (Chenn and McConnell, 1995; Chenn et al., 1998; Zhong et al., 1996; reviewed in Lu et al., 2000a,b). How a molecule

11 L. Sommer, M. Rao / Progress in Neurobiology 66 (2002) is partitioned into separate daughters is another subject of investigation. While few data are available in neural stem cells on how asymmetrical distribution of molecules is established relative to the axis of division, evidence from other species suggests that upregulation of synthesis in particular compartments as well as degradation in unwanted regions contribute to asymmetric distribution. For example, the asymmetrical distribution of Pie-1, a nuclear protein, that is localized to the germline blastomeres in C. elegans is achieved by enrichment and degradation (Reese et al., 2000). In Drosophila, the inscuteable protein is a central component of such a machinery. Within dividing Drosophila neural precursor cells, inscuteable directs both the orientation of the mitotic spindle and the asymmetric segregation of the proteins Numb, Prospero, Staufen, and Miranda (reviewed in Broadus and Doe, 1997; Knoblich, 1997). The identification of homologs of these proteins in other species has suggested that this machinery might be conserved from Drosophila to vertebrates. In this context it is intriguing to note that presenilins, identified on the basis of their role in the etiology of Alzheimers, have now been shown to also regulate Notch proteolytic processing (see, for example, Berezovska et al., 2000; Zhang et al., 2000). Loss of function phenotypes in Drosophila and in mammalian cells show reduced proteolytic release of the biologically active Notch intracellular domain and reduced Notch signaling (see, for example, Struhl and Adachi, 2000). Indeed, loss of function mutations of presenilin-1 have a embryonic lethal phenotype that affects multiple tissues and this phenotype resembles the loss of function phenotype seen in Notch mutants (Donoviel et al., 1999). A direct effect of presenilin on altering differentiation of neural stem cells has been reported (Handler et al., 2000). Neuroepithelial stem cells cultured from the ps1 ( / ) embryos show a decreased self-renewal capacity and premature differentiation (Hitoshi et al., 2000, Society of Neuroscience Abstract 23.7). Whether presenilin activity promotes asymmetric distribution of Notch, however, remains to be determined. Intracellular segregation of molecules has to be coupled to cytoskeletal organization, cytoplasmic segregation and mitotic orientation, and these movements have to be coordinated around an appropriate axis of division (reviewed in Lin and Schagat, 1997). Much of our understanding of this process comes from studying early events in the fertilized egg (Schatten et al., 2000a,b,c; Ciemerych et al., 2000). It is likely that the same regulatory processes will be involved in divisions in stem cells although this remains to be proven. Overall the data suggest that specifying whether a stem cell will undergo symmetric or asymmetric divisions is an important mechanism for regulating differentiation. Notch and Numb are likely important molecules that regulate the process of differentiation and their localization is subject to rigorous control. Whether Notch and Numb signaling plays a role in specifying asymmetric cell divisions in more differentiated cells is open to speculation. Both neuronal and glial precursors express these molecules and at least Numb Fig. 5. Numb localization division. Immunocytochemcal localization of Numb (red) in stem cells and precursor cells. Arrows point to example of asymmetric localization. Note the asymmetric cytoplasmic distribution of Numb in both multipotent neural stem cells (NEP) and more restricted precursor cells (NRP-neuron restricted precursors and GRP-glial restricted precursors). Photographs courtesy: Yuan Yuan Wu, University of Utah. appears to be asymmetrically localized (Fig. 5). However, it is not clear if these precursors undergo asymmetrical divisions or merely several cycles of symmetric divisions to provide a limited amplification of cell number. The availability of conditional knockouts (reviewed in Sauer, 1998) may allow a resolution of this question. 8. Signal integration by stem cells We have discussed how proliferative, apoptotic, and differentiation signals can act to regulate stem cell number and have shown how each specific set of signals can act at specific stages to regulate stem cell behavior. It is important to emphasize, however, that the particular decision a stem cell makes, be it to remain quiescent, proliferate, differentiate or die, represents the combinatorial action of multiple environmental signals. The sum total of these signals is assumed to constitute the stem cell compartment, or niche (reviewed in Watt and Hogan, 2000). Thus, stem cell behavior in such niches might be specified by context-dependent integration of cell extrinsic and cell intrinsic signals (Fig. 6A). In support of this view, it has recently been shown that during

12 12 L. Sommer, M. Rao / Progress in Neurobiology 66 (2002) 1 18 Fig. 6. (A) Integration of signals. Survival, proliferation, and differentiation in stem cells are controlled by the combinatorial action of multiple environmental signals. These signals include secreted factors acting at a long- or short-range, factors presented by the extracellular matrix or by neighboring cells, and signals transmitted via gap junctions. Furthermore, such extracellular signals are involved in an interplay with intracellular cues. Thus, integration of a context-specific set of multiple signal transduction pathways specifies the fate of a multipotent stem cell. (B) Community effects as an example of signal integration. TGF signaling elicits three alternative responses in neural crest-derived multipotent progenitor cells, dependent on the context and on ligand concentration. In isolated individual cells, TGF promotes a non-neural, smooth muscle-like fate; in the context of cells aggregating in a community, it acts either neurogenic at low factor concentrations or proapoptotic at slightly higher doses. Thus, so-called community effects provided by short-range cell cell interactions, can alter the biological activity of an instructive growth factor. These community effects are stage-specific since progenitor cells undergoing neuronal differentiation lose the competence to respond to the proapoptotic activity of TGF (Hagedorn et al., 1999, 2000). Drosophila eye development unique cell fates in multipotent progenitor cells are specified by the combinatorial activity of a small number of signaling pathways (Flores et al., 2000). Likewise, glial fate specification in the mouse PNS involves the integration of signals provided by the transcription factor Sox10 and multiple extracellular cues (Paratore et al., 2001). Such processes likely involve the cross-talk between distinct signal transduction pathways that might inhibit, potentiate or modulate each other to elicit specific biological responses in multipotent stem cells. The cues engaged in combinatorial signaling might be provided as secreted molecules, by the extracellular matrix, and by direct cell cell contact. Several examples of cross-talk between signaling pathways have been described on the molecular level (see e.g. Uusitalo et al., 1999; Whitman, 1998), but the impact of such cross-talk on cellular behavior, particularly in the vertebrate nervous system, is poorly understood. In Xenopus, two-step signaling by BMP factors and Wnt is involved in neural crest induction (LaBonne and Bronner-Fraser, 1998). Wnt signaling might also influence Notch-dependent fate decisions given that Wnt and Notch signaling share downstream signal transduction components

13 L. Sommer, M. Rao / Progress in Neurobiology 66 (2002) (reviewed in Uusitalo et al., 1999). In the nervous system, FGF-2 appears to cooperate with locally produced cytostatin C to stimulate proliferation and neurogenesis in neural stem cells, suggesting that broadly expressed multifunctional factors acquire specific activity by their interaction with local autocrine or paracrine factors (Taupin et al., 2000). Spatiotemporal regulation of developmental signaling often involves feedback from differentiating cells to progenitors (Freeman, 2000). Purkinje cells of the developing cerebellum produce Shh to stimulate the expansion of granule cell precursors (Wechsler-Reya and Scott, 1999). In the olfactory epithelium, differentiated olfactory neurons prevent proliferation and neuronal differentiation of their progenitors (Calof et al., 1998). Similarly, neuron-derived TGF appears to control both neurogenesis and the number of neurons produced from neural crest-derived multipotent progenitor cells (Hagedorn et al., 2000). However, signal integration has not only to be achieved by single cells but apparently also occurs across cell populations. Stem cells that share a common space and, thus, encounter the same environmental cues, display synchronized proliferation behavior and maintain a neighbor-to-neighbor relationship in the developing neuroepithelium (Cai et al., 1997b). Synchrony and contiguity is presumably achieved by gap junctions, but direct cell cell contact via transmembrane receptor molecules and local accumulation of secreted signals might also be involved. Short-range cellular interactions seem to affect other stem cell fates in addition to proliferation capacity. The Fas death receptor is broadly expressed in dissociated cultured embryonic neuroblasts while its spatiotemporal expression pattern is more restricted in situ, suggesting that environmental cues modulate Fas expression and the Fas-dependent death pathway (Cheema et al., 1999). Moreover, in the complex environment of an embryonic brain, neural progenitor cells deficient for the Rb gene undergo PCD while isolated mutant progenitor cells survive and differentiate in culture (Callaghan et al., 1999). Recently, it has been demonstrated that short-range cell cell interactions termed community effects (Gurdon et al., 1993) alter the biological response of multipotent progenitor cells to instructive growth factors such as TGF (Hagedorn et al., 1999, 2000) (Fig. 6B). Individual progenitor cells derived from the neural crest give rise to non-neural cells in the presence of TGF factors, whereas TGF -treated progenitor communities undergo neurogenesis and at moderately higher ligand doses cell death at the expense of the non-neural fate. Thus, TGF is able to induce three different fates in multipotent progenitor cells in a context-dependent manner. Thereby, cells within communities behave as a homogenous population with respect to their response, suggesting that mechanisms exist to coordinate progenitor cell behavior across the population (Hagedorn et al., 1999, 2000). Similar to neural crest-derived progenitor cells, a non-neural fate can be suppressed in cortical stem cells by cell contact (Tsai and McKay, 2000). This effect might depend on membrane-bound signals as it can be mimicked by treatment of stem cells with membrane extract. In sum, the genetic program of a stem cell appears to be defined by multiple signaling pathways that constitute a site-specific signaling network. The dynamic nature of this signaling network allows precise control of the multiple fates of the stem cell population by changes in the individual signaling components. It will be a challenge to identify all the components involved and to determine how their composite signaling activity is integrated by stem cells. The concept of signaling networks acting on stem cells also implies that, in order to study stem cell behavior, careful attention needs to be paid to the context of the experiment. Cell density, factor concentration, and the time of isolation may all affect the outcome of any perturbation experiments. In the future, functional genomics and proteomics appear likely to become invaluable techniques to elucidate the signaling networks as a whole. 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