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Neuronal potential and lineage determination by neural stem cells
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Neuronal potential and lineage determination by neural stem cells Sean J Morrison How do neural stem cells ensure that they give rise to the right number and type of neurons at the right time? Over the past year several regulatory mechanisms have been identified, including promotion of neurogenesis by proneural bHLH genes, instruction of gliogenesis by Notch, and cell-intrinsic changes in the neurogenic capacity of stem cells in culture and in vivo. Addresses Howard Hughes Medical Institute, Departments of Internal Medicine and Cell and Developmental Biology, 3215 CCGC, University of Michigan, Ann Arbor, MI 48109-0934, USA; e-mail: [email protected] Current Opinion in Cell Biology 2001, 13:666–672 0955-0674/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations bHLH basic helix–loop–helix (transcription factor) BMP bone morphogenic protein CBP CREB binding protein CNS central nervous system mNCSC migrating NCSC NCSC neural crest stem cell Nrg-1 neuregulin PNS peripheral nervous system snNCSC sciatic nerve NCSC
Introduction Neural stem cells are self-renewing, multipotent progenitors that give rise to the diverse types of neurons and glia that compose the nervous system. There are two main types of neural stem cells, central nervous system (CNS) stem cells and neural crest stem cells (NCSCs). CNS stem cells are defined by their ability to give rise to neurons, astrocytes and oligodendrocytes [1–3]. NCSCs are defined by their ability to give rise to the neurons, and glia of the peripheral nervous system (PNS) as well as other connective cell types like smooth muscle [4–6]. Also, different subtypes of CNS stem cells and NCSCs can be identified at different times of development or in different regions of the CNS or PNS, indicating considerable diversity within these categories of neural stem cells. For example, functionally distinct stem cell populations have been cultured from each of the major subdivisions of the developing CNS, as well as from the retina [7], forebrain [8,9], hippocampus [10] and spinal cord [11] of the adult CNS (reviewed in [12]). Each of these types of stem cells gives rise to different types of cells in vivo. More is known about lineage determination in neural stem cells than in any other mammalian stem cell system. This is because unlike many types of stem cells (such as from the gut epithelium or hematopoietic system), neural stem cells undergo self-renewal and multilineage differentiation in culture. Moreover, the survival, proliferation,
and differentiation of individual stem cell colonies can be monitored simultaneously in the presence of different growth factors in culture, allowing us to distinguish between the instructive and selective effects of growth factors. This provides the opportunity to study how individual growth factors influence the process of lineage determination. In NCSCs, bone morphogenic proteins (BMPs) instruct neuronal differentiation, neuregulin (Nrg-1; also known as glial growth factor) instructs glial differentiation, and transforming growth factor-β instructs myofibroblast differentiation [5,13]. Similar observations were made in CNS stem cells, in which BMPs sometimes instruct neurogenesis, ciliary neurotrophic factor or BMPs instruct astrocytic differentiation, and thyroid hormone instructs oligodendrocyte differentiation [14–16]. The next step will be to study how the responses to different lineage determination factors are coordinated such that neural stem cells undergo multilineage differentiation in a way that is developmentally appropriate. Recent studies have focused on different aspects of the regulation of neurogenesis, from neuronal potential, to neurogenic factor responsiveness, to reinforcing neuronal lineage determination, to terminating neurogenesis. Findings from these studies indicate that we have to be careful about making conclusions regarding the neuronal potential of progenitors from the CNS, as oligodendrocyte precursor cells (glial progenitors that do not initially respond to neurogenic factors in culture) can acquire neuronal potential and CNS stem cell properties over time in culture [17••]. With regard to the response of neural stem cells to neurogenic factors, it has been demonstrated that neural stem cells undergo changes over time in vivo in their neurogenic properties while remaining multipotent and self-renewing. Cell-intrinsic changes in responsiveness to neurogenic signals, such as BMPs, influence the number and type of neurons that stem cells can make in vivo [18•,19•]. To reinforce neurogenesis, proneural basic helix–loop–helix (bHLH) transcription factors not only drive neurogenesis by activating the expression of a cascade of neuronal genes, but they inhibit the expression of glial genes [20,21•,22••]. Finally, if proneural genes inhibit gliogenesis, how is neurogenesis terminated so that gliogenesis can begin? Part of the answer is that Notch activation acts in neural stem cells as a switch that terminates neurogenesis and initiates gliogenesis, even in the continued presence of neurogenic growth factors [23•,24•,25••,26•,27•]. Each of these recent studies on different aspects of neural stem cell differentiation has implications for our understanding of important principles of the regulation of neurogenesis.
The neuronal potential of neural progenitors In the neural stem cell literature, the results from studies using cultured progenitors are strikingly inconsistent with
Neuronal potential and lineage determination by neural stem cells Morrison
those from studies using uncultured progenitors. Studies of cultured cells have emphasized the remarkable ability of stem cells from one region of the nervous system to give rise to neurons found in other regions [28–31]. This led to the idea of a common CNS stem cell with the potential to give rise to all types of neurons in the CNS. In contrast, studies of uncultured progenitors transplanted to a different region of the nervous system or to an earlier time in development have consistently observed restrictions in the ability of these stem cells to form the neurons that would normally form at these other places or times [32–35]. It seems unlikely that the higher purity of multipotent progenitors in the cultured CNS cell preparations (cultures favor the proliferation and survival of multipotent progenitors) were responsible for this difference. This is because, even when NCSCs are prospectively identified and purified by flow-cytometry from uncultured tissue, transplantation of these uncultured NCSCs in vivo reveals restrictions or biases in neuronal potential [19•]. Instead, it seems likely that neural progenitors may acquire a broader developmental potential as a result of proliferation in culture. The evidence that developmental potential can sometimes broaden in culture is presented in detail in an excellent recent review [36]. The most direct evidence that CNS progenitors can acquire a broader developmental potential comes from studies of oligodendrocyte precursor cells. After decades of studies in vitro and in vivo, these cells were thought to be glial committed, but recently were shown to acquire the ability not only to make neurons but to form neurospheres (CNS stem cell colonies) after being cultured in a series of growth factors [17••]. Indeed, it is possible to culture neurospheres from any region of the adult CNS, including regions that show no evidence of neurogenic activity in vivo [7,37]. One possible interpretation of this is that glial progenitors that lack neurogenic potential or multipotency in vivo may acquire the ability to make neurons and stem cell colonies in culture [38,39]. It will be important to determine whether the expression of patterning genes that specify region-specific neuronal fates in vivo is lost in culture and whether the loss of expression of such patterning genes leads progenitors to acquire a broader developmental potential in culture than they would have in vivo. To resolve these issues it will be necessary to continue to prospectively identify and purify neural stem cells [6,40,41] so that their properties can be studied in vivo as well as in vitro.
Changes in growth factor sensitivity regulate neuronal lineage determination How does a neural stem cell ensure that it produces the right number and kind of neurons? Do changes in the expression of neurogenic signals in the environment modulate the number and type of neurons produced? Or do cell-intrinsic changes in neural stem cells affect the way stem cells respond to neurogenic factors in the environment? Both types of mechanisms are likely to be involved, but work published over the past year has emphasized that
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neural stem cells change their cell-intrinsic properties over time in a way that influences the number and type of neurons they can generate. Hematopoietic stem cells [42–44], retinal stem cells [45], and cortical progenitors [46] have been demonstrated to undergo cell-intrinsic changes in developmental or neuronal subtype potential over time. Nonetheless, the literature on CNS stem cells has tended to emphasize the ways in which stem cells retain a broad neuronal potential rather than evidence of restrictions in neuronal potential. CNS stem cells undergo restrictions that reduce the number and types of neurons they make at later developmental stages. CNS stem cells from the postnatal subventricular zone failed to form large projection neurons or neurons in the cortex or hippocampus upon transplantation into the lateral ventricles of E15 fetuses [47]. This suggests that subventricular zone stem cells, which normally give rise to interneurons of the olfactory bulb, may have lost the potential to make projection neurons and certain other types of neurons that form in other regions of the CNS. A more recent paper by Temple and colleagues [18•] shows that the neurogenic properties of cortical stem cells undergo cell-intrinsic changes even over much shorter developmental times. Temple and colleagues [18•] isolated stem cells from the embryonic cortical ventricular zone and monitored the proliferation and differentiation of individual stem cells in culture. They found that the stem cells always made neurons first and glia second, just as they do in vivo. This suggested that the information required for the generation of neurons followed by glia was intrinsic to an isolated clone of stem cells and their progeny. Even more interesting was the observation that stem cells isolated from later embryonic stages gave rise to fewer neurons before initiating gliogenesis than stem cells isolated at earlier stages. This again mirrored what is observed in vivo. The molecular basis for this developmental change in neurogenic capacity was not determined but could relate to cell-intrinsic changes in sensitivity to lineage determination factors (reviewed in [48]). Within the PNS, analogous changes occur in NCSCs over time. Early migrating NCSCs (mNCSCs) can be isolated from E10.5 neural tube explants [4] while postmigratory NCSCs can be isolated from E14.5 sciatic nerves (snNCSCs) [6]. The neurogenic potentials of these two NCSC populations were compared by transplanting uncultured, purified rat NCSCs from either stage of development into the neural crest migration pathway of chick embryos [19•,49]. In this assay the rat NCSCs migrated along with chick neural crest cells and differentiated into diverse derivatives throughout the chick PNS. Both the mNCSCs and the snNCSCs gave rise to cholinergic neurons in parasympathetic ganglia, noradrenergic neurons in sympathetic ganglia, and glia in many locations, as would be expected; however, two differences were observed [19•]. The snNCSCs consistently gave rise to fewer neurons than the mNCSCs, and the snNCSCs only rarely gave rise to noradrenergic neurons in vivo, despite the fact that they
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Figure 1
Delta CNTF
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readily gave rise to noradrenergic neurons in culture [50]. The snNCSCs also required 50-fold higher levels of BMP stimulation than the mNCSCs to initiate neurogenesis. As BMPs drive autonomic neurogenesis and higher levels of BMP stimulation were required for noradrenergic differentiation [19•], the data suggest that the decreased sensitivity of the snNCSCs led to a quantitative reduction in neurogenic capacity and a bias against noradrenergic differentiation in the presence of limiting BMP levels in vivo. Thus, just as with cortical CNS stem cells, NCSCs exhibit reduced neurogenic capacity with increasing developmental time and biases that reduce their ability to produce certain types of neurons, all while remaining multipotent and self-renewing. Changes in growth factor sensitivity over developmental time may be a general mechanism by which stem cells modulate their developmental potential and differentiation. It will also be interesting to determine whether changes in the sensitivity to neurogenic factors are behind the increased neurogenic potential of CNS progenitors in culture [17••,39].
Proneural bHLH genes inhibit gliogenesis Neurogenic factors promote neurogenesis by inducing the expression of proneural bHLH transcription factors such as Neurogenin and Mash-1. These proneural bHLH proteins are master regulators of neuronal differentiation that coordinate the expression of neuronal genes. For example, BMPs promote autonomic neurogenesis in the PNS by inducing the expression of Mash-1 in NCSCs [13]. Mash-1 then turns on a cascade of genes that collectively confer
Proneural genes such as Neurogenin1 promote neurogenesis by forming complexes with the CBP–p300–Smad1 transcriptional coactivator, binding to the promoters of neuronal genes, and activating transcription. Activation of the LIF receptor by CNTF promotes gliogenesis by activating STAT1/3. Activated STAT1/3 can also form a complex with the CBP–p300–Smad1 transcriptional coactivator, and bind to the promoters of glial genes, activating their transcription. Neurogenin1, and perhaps other proneural genes, can inhibit glial differentiation by sequestering the CBP–p300–Smad1 transcriptional coactivator to neuronal promoters and away from glial promoters, as well as by inhibiting STAT activation [22••]. BMPs can promote either neuronal differentiation or glial differentiation by activating Smad1, which binds to CBP–p300. In some cells, BMPs might also promote neuronal differentiation by promoting expression of proneural bHLH genes. Notch promotes gliogenesis and inhibits neurogenesis [23•,24•,25••,26•,27•]. It may do this by inhibiting the expression of proneural bHLH transcription factors and perhaps by promoting the transcription of glial genes by other mechanisms.
both pan-neuronal and subtype-specific aspects of autonomic neuronal identity [51]. Other types of proneural bHLH genes, like Neurogenin1 [52], promote differentiation into other types of neurons in the PNS and CNS. Three groups recently published evidence indicating that, in addition to acting as master regulators of neurogenesis, proneural bHLH transcription factors also inhibit gliogenesis [20,21•,22••]. Deficiencies in various combinations of proneural genes led not only to a loss of certain types of neurons, but to premature gliogenesis as well [20,21•]. One mechanism by which proneural genes might inhibit gliogenesis is by the sequestration of transcriptional coactivators away from glial promoters, preventing the expression of glial genes. Sun, Greenberg and colleagues [22••] presented evidence that Neurogenin1 can bind the CBP–Smad1 (CREB binding protein) transcription complex, preventing it from being recruited to glial promoters (Figure 1). By sequestering the CBP–Smad1 complex at neuronal promoters, proneural bHLH genes may both promote the expression of neuronal genes and inhibit the expression of glial genes. These observations resonate with the earlier discovery that transcription factors that regulate myeloerythroid differentiation in the hematopoietic system consistently act by promoting the acquisition of one fate while inhibiting the acquisition of alternative fates [53]. Thus, it may be a general strategy for lineagedetermining transcription factors to reinforce the process of differentiation by both promoting one fate and inhibiting alternative fates.
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Notch activation promotes gliogenesis Throughout the nervous system, neurogenesis occurs first and is followed by gliogenesis. How do neural stem cells terminate neurogenesis and initiate gliogenesis? Do the neurogenic signals just go away, or is neurogenesis actively shut down when gliogenesis begins? The expression of BMP2 or BMP4 near forming autonomic ganglia instructs NCSCs to differentiate into autonomic neurons [13,54–56]. But instead of turning off when it is time for gliogenesis to begin, BMP expression persists. So how does the second wave of neural crest progenitors undergo gliogenesis with the continued presence of the strongly neurogenic BMPs? An obvious possibility is a feedback signal from the neurons that would overcome the neurogenic influence of BMPs and promote gliogenesis. Indeed, the nascent neurons do express the gliogenic factor Nrg-1, but when NCSCs are simultaneously exposed to BMP2 and Nrg-1, neurogenesis continues and gliogenesis is completely suppressed [57]. Thus, Nrg-1 cannot explain the switch to gliogenesis. It was demonstrated recently that Notch activation in NCSCs instructs glial differentiation in a way that is dominant to the neurogenic influence of BMP2 and BMP4 [25••]. Furthermore, Notch activation caused glial lineage determination much more quickly than is observed in response to Nrg-1. Exposure to a soluble form of a Notch ligand (Delta) for only 24 hours caused an irreversible loss of neuronal potential, such that a subsequent 4 days of culture in BMPs resulted in continued glial differentiation, rather than neuronal differentiation. The transient expression of Notch ligands by nascent autonomic neurons may thus trigger a switch in NCSCs to terminate neuronal differentiation and initiate gliogenesis, despite the continued presence of BMPs. Is Notch activation a signal for the termination of neurogenesis and the initiation of gliogenesis throughout the nervous system? Notch pathway activation has also been observed to promote the acquisition of glial fates in vivo in the postnatal retina [23•] and in the fetal telencephalon [24•]. It was not clear in these cases whether Notch was instructing gliogenesis or just inhibiting neurogenesis, but in other studies of retinal neurogenesis in vivo, Notch pathway activation instructed the generation of Muller glia at the expense of neurons [26•,58]. Notch activation also instructs astrocytic differentiation by CNS stem cells from the adult hippocampus [27•]. Although there are several examples of cases in which Notch activation promoted gliogenesis by neural stem cells, there are also examples when Notch signaling was necessary or sufficient to maintain neural stem cells in an undifferentiated state [59,60]. Overall, it would appear that Notch signaling maintains multipotency in some neural stem cells but promotes glial differentiation in others or at other times during development. Another interpretation is that Notch activation may cause overt glial differentiation in some stem cells, whereas in other cases it may cause only glial lineage determination, with other factors additionally required for overt differentiation. One trend that may be emerging from the literature is that at early times of
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development, prior to the onset of gliogenesis, Notch activation may promote a maintenance of multipotentiality, but at later times during development, when gliogenesis has started or is about to start, it may promote gliogenesis. In this way, Notch activation would be modulated to have an effect that was appropriate to developmental stage. What is the molecular mechanism by which Notch promotes gliogenesis? As Notch inhibits the expression of Neurogenin, Mash-1, and other proneural bHLH genes [25••,52], it would be expected to promote gliogenesis by inhibiting the ability of proneural genes to inhibit gliogenesis (Figure 1; reviewed in [61]). Notch may also have other more direct mechanisms for promoting expression of glial genes. For example, Notch remained able to slightly promote expression of the glial marker GFAP (glial fibrillary acidic protein) even after mutation of the STAT3 (signal transducers and activators of transcription) binding site in the GFAP promoter [27•]. As the STAT3 binding site is where the STAT3–CBP–Smad1 complex activates transcription, this implies that part of the mechanism by which Notch promotes gliogenesis is independent of the CBP–Smad1 complex and, therefore, independent of the ability of proneural genes to sequester this complex. Notch probably functions in many regions of the nervous system as a switch that terminates neurogenesis and causes glial lineage determination, in part by inhibiting expression of proneural bHLH factors.
Conclusions Our understanding of neuronal lineage determination has advanced during the past year with the demonstration that proneural genes inhibit gliogenesis [20,21•,22••], and that Notch activation instructs gliogenesis [25••,27•]. We have also learned that neuronal potential is a moving target. Stem cells can reduce their sensitivity to neurogenic factors over time in vivo, reducing their capacity to form neurons and biasing the types of neurons they can make [18•,19•]. Progenitors that lack the ability to respond to neurogenic signals can acquire neurogenic potential in culture [17••]. We must stop thinking of neurogenic capacity and neuronal potential in black and white terms. The neuronal potentials of progenitors in vivo exist in shades of gray, shades that can change over time in vivo, and shades that can change (perhaps unphysiologically) in culture.
Acknowledgements I am supported as an Assistant Investigator of the Howard Hughes Medical Institute and a Searle Scholar. I apologize to those authors whose work was not cited due to space limitations. I thank Nancy Joseph and Theodora Ross for helpful comments on the manuscript.
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Kondo T, Raff M: Oligodendrocyte precursor cells reprogrammed to become multipotential CNS stem cells. Science 2000, 289:1754-1757. Oligodendrocyte precursor cells were studied for many years by many laboratories in vitro and in vivo and were thought to be committed to glial fates. Prospectively identified and purified oligodendrocyte precursor cells were not initially able to respond to neurogenic signals in culture, but after exposure to BMP-containing medium followed by FGF-containing medium, these cells not only made neurons, but they formed neurospheres and exhibited the properties of neural stem cells. These data strongly suggest that CNS cells that are committed to glial fates in vivo can reprogram their developmental potential in culture. The ability of oligodendrocyte precursors to adopt the properties of CNS stem cells over time in culture emphasizes the need to prospectively identify CNS stem cells to prove that cells exist in vivo with properties that are similar to the self-renewing, multipotent cells that have been characterized in vitro. 18. Qian X, Shen Q, Goderie SK, He W, Capela A, Davis AA, Temple S: • Timing of CNS cell generation: a programmed sequence of neuron and glial cell production from isolated murine cortical stem cells. Neuron 2000, 28:69-80. A program that brings about neuronal differentiation before glial differentiation is encoded intrinsically within CNS stem cells. Individual cells from the E10 cortex were cultured at clonal density and then the proliferation and differentiation of individual clones were followed by time-lapse video microscopy. In doing so, Qian et al. retrospectively constructed a ‘family tree’ for each stem cell clone. Multipotent progenitors in culture always generated neuroblasts before glioblasts; furthermore, multipotent progenitors from the E10 cortex gave rise to many more neurons than did multipotent progenitors from the E16 cortex even though there was no obvious difference in their capacity to generate glia. Thus, in addition to encoding the order of neurogenesis/ gliogenesis, stem cells also change over time in their propensity to generate neurons, despite remaining multipotent.
19. White PM, Morrison SJ, Orimoto K, Kubu CJ, Verdi JM, Anderson DJ: • Neural crest stem cells undergo cell-intrinsic developmental changes in sensitivity to instructive differentiation signals. Neuron 2001, 29:57-71. Migrating mNCSCs were purified from E10.5 neural tube explants while postmigratory snNCSCs were purified by flow-cytometry from E14.5 sciatic nerves [6]. When these cells were injected into the neural crest migration pathways of chick embryos the rat NCSCs engrafted throughout the chick PNS. Both mNCSCs and snNCSCs were nearly pure populations of self-renewing multipotent progenitors that gave rise to large numbers of cholinergic and noradrenergic neurons in vitro. But in vivo the snNCSCs consistently gave rise to 10-fold fewer neurons, and rarely gave rise to noradrenergic neurons. The snNCSCs were 50-fold less sensitive to the neurogenic activity of BMPs and noradrenergic differentiation required higher levels of BMP stimulation. This suggested that levels of BMPs were limiting in vivo but not in vitro for neuronal differentiation and the acquisition of noradrenergic identity. Cell-intrinsic changes in growth factor sensitivity in stem cells in vivo lead to quantitative reductions in the capacity for neuronal differentiation and biases in neuronal subtype potential, even while the stem cells remain multipotent and self-renewing. To detect mechanisms like this that regulate stem cell differentiation, neural stem cells must be prospectively identified [6] and studied in vivo. 20. Tomita K, Moriyoshi K, Nakanishi S, Guillemot F, Kageyama R: Mammalian achaete-scute and atonal homologs regulate neuronal versus glial fate determination in the central nervous system. EMBO J 2000, 19:5460-5472. 21. Nieto M, Schuurmans C, Britz O, Guillemot F: Neural bHLH genes • control the neuronal versus glial fate decision in cortical progenitors. Neuron 2001, 29:401-413. Nieto et al. analyzed mice that were deficient for Mash1 and/or Neurogenin2 and concluded that, in addition to reduced neurogenesis, glial precursors were generated earlier and in greater numbers in the double mutant mice. However, only a minority of animals exhibited premature GFAP expression, suggesting that loss of Mash1 and Neurogenin2 may accelerate glial lineage determination but that other factors determine the timing of overt differentiation. The appearance of an increase in the number of immature glia in double mutants in vivo, and the increased glial lineage determination observed in one subset of progenitors from Mash-1 deficient mice, suggest that proneural genes are necessary to avoid premature glial lineage determination, but loss of proneural genes is not sufficient to cause glial commitment. By comparing the results of Tomita et al. [20] to the results of Nieto et al., it appears that gliogenesis is accelerated to different extents in different regions of the nervous system, depending on which proneural genes are deleted. 22. Sun Y, Nadal-Vicens M, Misono S, Lin MZ, Zubiaga A, Hua X, Fan G, •• Greenberg ME: Neurogenin promotes neurogenesis and inhibits glial differentiation by independent mechanisms. Cell 2001, 104:365-376. Sun et al. observed that Neurogenin1 strongly inhibited LIF-induced (leukemia inhibitory factor) glial differentiation by cortical progenitors in culture, independent of its ability to promote neuronal differentiation. Within the GFAP promoter, the STAT binding site was necessary for the full Neurogenin1-mediated inhibition of GFAP expression but Neurogenin1 binding sites were not. The STAT binding site promotes GFAP transcription by binding a complex of STAT1/3 (activated by LIF/CNTF [ciliary neurotrophic factor] signaling), CBP or p300 (ubiquitously expressed transcriptional coactivators), and Smad1 (activated by BMP signaling). Neurogenin1 inhibits the assembly of this complex by binding to both CBP and Smad1, interfering with the ability of CBP–Smad1 to bind STAT3 (Figure 1). Thus, Neurogenin1 may inhibit glial lineage determination by sequestering the CBP–p300–Smad1 transcriptional complex away from the promoters of glial genes. 23. Furukawa T, Mukherjee S, Bao ZZ, Morrow EM, Cepko CL: rax, • Hes1, and notch1 promote the formation of muller glia by postnatal retinal progenitor cells. Neuron 2000, 26:383-394. Constitutively active Notch1 and Hes1, a downstream mediator of the transcriptional effects of Notch signaling, were overexpressed in the retinas of P0 rats in vivo. Overexpression of either gene caused an increase in the expression of Muller glial markers. However, it was unclear in these experiments whether Notch signaling was instructing glial differentiation, or whether glial differentiation occurred by default in response to an inhibition of neuronal differentiation. 24. Gaiano N, Nye JS, Fishell G: Radial glial identity is promoted by • Notch1 signaling in the murine forebrain. Neuron 2000, 26:395-404. Cells in the mouse fetal forebrain (telencephalon) were infected with a retroviral vector bearing the constitutively active Notch1 intracellular domain. Cells that expressed constitutively active Notch acquired a radial glial identity and then became periventricular astrocytes postnatally. As many radial glia and periventricular astrocytes are thought to be multipotent, it is not clear in these experiments whether Notch is promoting glial differentiation or a maintenance of multipotentiality.
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25. Morrison SJ, Perez S, Verdi JM, Hicks C, Weinmaster G, Anderson DJ: •• Transient Notch activation initiates an irreversible switch from neurogenesis to gliogenesis by neural crest stem cells. Cell 2000, 101:499-510. Most models of Notch function in vertebrate progenitors hold that Notch acts reversibly to inhibit differentiation, maintaining progenitors in an undifferentiated state. In this study, Notch activation not only instructed glial differentiation but it caused an irreversible loss of neuronal potential in NCSCs. For example, NCSCs were exposed to Notch ligand for only 24 hours, then the Notch ligand was washed out of the culture medium and replaced with medium containing the neurogenic factor BMP2. Instead of undergoing neuronal differentiation, the NCSCs continued to undergo glial differentiation despite the loss of Notch ligand and the presence of BMP2. These observations suggest that Notch activation in snNCSCs sends a positive signal that promotes gliogenesis rather than just a reversible negative signal inhibiting neurogenesis. 26. Hojo M, Ohtsuka T, Hashimoto N, Gradwohl G, Guillemot F, • Kageyama R: Glial cell fate specification modulated by the bHLH gene Hes5 in mouse retina. Development 2000, 127:2515-2522. Hes5, a downstream mediator of the transcriptional effects of Notch signaling, was necessary and sufficient to increase the numbers of Muller glia in the E15-P1 mouse retina. Because expression of Hes5 did not promote proliferation or cell death, and increased the numbers of glia while decreasing the number of neurons, it appeared that Hes5 instructed glial differentiation. Kageyama and colleagues, studies of retinal development suggest that while Hes5 promotes glial differentiation, Hes1 may promote a maintenance of multipotentiality. This suggests that there may be a bifurcation within the Notch pathway as different downstream mediators of Notch signaling may have different effects on progenitor differentiation. 27. •
Tanigaki K, Nogaki F, Takahashi J, Tashiro K, Kurooka H, Honjo T: Notch1 and Notch3 instructively restrict bFGF-responsive multipotent neural progenitor cells to an astroglial fate. Neuron 2001, 29:45-55. Constitutively active Notch instructed glial differentiation in stem cells from the adult hippocampus and even transient Notch activation was sufficient to cause glial lineage determination. Notch signaling slightly promoted GFAP expression, even in the absence of the gliogenic factor CNTF, and this promotion did not require STAT3 activation or even the STAT binding site in the GFAP promoter. This suggests that Notch’s promotion of gliogenesis does not depend on signaling through the STAT pathway and is not entirely based on an inhibition of Neurogenin’s sequestration of the CBP–p300–Smad1 complex from the STAT binding site [22••]. Thus, Notch may promote gliogenesis in additional ways beyond the inhibition of proneural gene expression. 28. Suhonen JA, Peterson DA, Ray J, Gage FH: Differentiation of adult hippocampus-derived progenitors into olfactory neurons in vivo. Nature 1996, 383:624-627. 29. Brustle O, Spiro AC, Karram K, Choudhary K, Okabe S, McKay RDG: In vitro-generated neural precursors participate in mammalian brain development. Proc Natl Acad Sci USA 1997, 94:14809-14814. 30. Fricker RA, Carpenter MK, Winkler C, Greco C, Gates MA, Bjorklund A: Site-specific migration and neuronal differentiation of human neural progenitor cells after transplantation in the adult rat brain. J Neurosci 1999, 19: 5990-6005. 31. Shihabuddin LS, Horner PJ, Ray J, Gage FH: Adult spinal cord stem cells generate neurons after transplantation in the adult dentate gyrus. J Neurosci 2000, 20:8727-8735. 32. LeDouarin NM: Cell line segregation during peripheral nervous system ontogeny. Science 1986, 231:1515-1522. 33. Campbell K, Olsson M, Bjorklund A: Regional incorporation and site-specific differentiation of striatal precursors transplanted to the embryonic forebrain ventricle. Neuron 1995, 15:1259-1273. 34. Na E, McCarthy M, Neyt C, Lai E, Fishell G: Telencephalic progenitors maintain anteroposterior identities cell autonomously. Curr Biol 1998, 8:987-990. 35. Olsson M, Bjerregaard K, Winkler C, Gates M, Bjorklund A, Campbell K: Incorporation of mouse neural progenitors transplanted into the rat embryonic forebrain is developmentally regulated and dependent upon regional adhesive properties. Eur J Neurosci 1998, 10:71-85. 36. Anderson DJ: Stem cells and pattern formation in the nervous system: the possible versus the actual. Neuron 2001, 30:19-35.
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38. Gage FH: Stem cells of the central nervous system. Curr Opin Neurobiol 1998, 8:671-676. 39. Palmer TD, Markakis EA, Willhoite AR, Safar F, Gage FH: Fibroblast growth factor-2 activates a latent neurogenic program in neural stem cells from diverse regions of the adult CNS. J Neurosci 1999, 19:8487-8497. 40. Uchida N, Buck DW, He D, Reitsma MJ, Masek M, Phan TV, Tsukamoto AS, Gage FH, Weissman IL: Direct isolation of human central nervous system stem cells. Proc Natl Acad Sci USA 2000, 97:14720-14725. 41. Kawaguchi A et al.: Nestin-EGFP transgenic mice: visualization of the self-renewal and multipotency of CNS stem cells. Mol Cell Neurosci 2001, 17:259-273. 42. Ikuta K, Kina T, Macneil I, Uchida N, Peault B, Chien YH, Weissman IL: A developmental switch in thymic lymphocyte maturation potential occurs at the level of hematopoietic stem cells. Cell 1990, 62:863-874. 43. Kantor AB, Stall AM, Adams S, Herzenberg LA, Herzenberg LA: Differential development of progenitor activity for three B-cell lineages. Proc Natl Acad Sci USA 1992, 89:3320-3324. 44. Morrison SJ, Hemmati HD, Wandycz AM, Weissman IL: The purification and characterization of fetal liver hematopoietic stem cells. Proc Natl Acad Sci USA 1995, 92:10302-10306. 45. Cepko CL: The roles of intrinsic and extrinsic cues and bHLH genes in the determination of retinal cell fates. Curr Opin Neurobiol 1999, 9:37-46. 46. McConnell SK: Strategies for the generation of neuronal diversity in the developing central nervous system. J Neurosci 1995, 15:6987-6998. 47.
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48. Morrison SJ: The last shall not be first: the ordered generation of progeny from stem cells. Neuron 2000, 28:1-3. 49. White PA, Anderson DJ: In vivo transplantation of mammalian neural crest cells into chick hosts reveals a new autonomic sublineage restriction. Development 1999, 126:4351-4363. 50. Morrison SJ, Csete M, Groves AK, Melega W, Wold B, Anderson DJ: Culture in reduced levels of oxygen promotes clonogenic sympathoadrenal differentiation by isolated neural crest stem cells. J Neurosci 2000, 20:7370-7376. 51. Lo L, Tiveron MC, Anderson DJ: MASH1 activates expression of the paired homeodomain transcription factor Phox2a, and couples pan-neuronal and subtype-specific components of autonomic neuronal identity. Development 1998, 125:609-620. 52. Ma Q, Chen ZF, Barrantes IB, de la Pompa JL, Anderson DJ: Neurogenin 1 is essential for the determination of neuronal precursors for proximal cranial sensory ganglia. Neuron 1998, 20:469-482. 53. Orkin SH: Diversification of haematopoietic stem cells to specific lineages. Nat Rev Genet 2000, 1:57-64. 54. Reissman E, Ernsberger U, Francis-West PH, Rueger D, Brickell PD, Rohrer H: Involvement of bone morphogenetic protein-4 and bone morphogenetic protein-7 in the differentiation of the adrenergic phenotype in developing sympathetic neurons. Development 1996, 122:2079-2088. 55. Schneider C, Wicht H, Enderich J, Wegner M, Rohrer H: Bone morphogenetic proteins are required in vivo for the generation of sympathetic neurons. Neuron 1999, 24:861-870. 56. Pisano JM, Colon-Hastings F, Birren SJ: Postmigratory enteric and sympathetic neural precursors share common, developmentally regulated, responses to BMP2. Dev Biol 2000, 227:1-11. 57.
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60. Nakamura Y, Sakakibara SI, Miyata T, Ogawa M, Shimazaki T, Weiss S, Kageyama R, Okano H: The bHLH gene Hes1 as a repressor of the neuronal commitment of CNS stem cells. J Neurosci 2000, 20:283-293. 61. Morrison SJ: Neuronal differentiation: proneural genes inhibit gliogenesis. Curr Biol 2001, 11:R349-R351.
Neuronal potential and lineage determination by neural stem cells Sean J Morrison How do neural stem cells ensure that they give rise to the right number and type of neurons at the right time? Over the past year several regulatory mechanisms have been identified, including promotion of neurogenesis by proneural bHLH genes, instruction of gliogenesis by Notch, and cell-intrinsic changes in the neurogenic capacity of stem cells in culture and in vivo. Addresses Howard Hughes Medical Institute, Departments of Internal Medicine and Cell and Developmental Biology, 3215 CCGC, University of Michigan, Ann Arbor, MI 48109-0934, USA; e-mail: [email protected] Current Opinion in Cell Biology 2001, 13:666–672 0955-0674/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations bHLH basic helix–loop–helix (transcription factor) BMP bone morphogenic protein CBP CREB binding protein CNS central nervous system mNCSC migrating NCSC NCSC neural crest stem cell Nrg-1 neuregulin PNS peripheral nervous system snNCSC sciatic nerve NCSC
Introduction Neural stem cells are self-renewing, multipotent progenitors that give rise to the diverse types of neurons and glia that compose the nervous system. There are two main types of neural stem cells, central nervous system (CNS) stem cells and neural crest stem cells (NCSCs). CNS stem cells are defined by their ability to give rise to neurons, astrocytes and oligodendrocytes [1–3]. NCSCs are defined by their ability to give rise to the neurons, and glia of the peripheral nervous system (PNS) as well as other connective cell types like smooth muscle [4–6]. Also, different subtypes of CNS stem cells and NCSCs can be identified at different times of development or in different regions of the CNS or PNS, indicating considerable diversity within these categories of neural stem cells. For example, functionally distinct stem cell populations have been cultured from each of the major subdivisions of the developing CNS, as well as from the retina [7], forebrain [8,9], hippocampus [10] and spinal cord [11] of the adult CNS (reviewed in [12]). Each of these types of stem cells gives rise to different types of cells in vivo. More is known about lineage determination in neural stem cells than in any other mammalian stem cell system. This is because unlike many types of stem cells (such as from the gut epithelium or hematopoietic system), neural stem cells undergo self-renewal and multilineage differentiation in culture. Moreover, the survival, proliferation,
and differentiation of individual stem cell colonies can be monitored simultaneously in the presence of different growth factors in culture, allowing us to distinguish between the instructive and selective effects of growth factors. This provides the opportunity to study how individual growth factors influence the process of lineage determination. In NCSCs, bone morphogenic proteins (BMPs) instruct neuronal differentiation, neuregulin (Nrg-1; also known as glial growth factor) instructs glial differentiation, and transforming growth factor-β instructs myofibroblast differentiation [5,13]. Similar observations were made in CNS stem cells, in which BMPs sometimes instruct neurogenesis, ciliary neurotrophic factor or BMPs instruct astrocytic differentiation, and thyroid hormone instructs oligodendrocyte differentiation [14–16]. The next step will be to study how the responses to different lineage determination factors are coordinated such that neural stem cells undergo multilineage differentiation in a way that is developmentally appropriate. Recent studies have focused on different aspects of the regulation of neurogenesis, from neuronal potential, to neurogenic factor responsiveness, to reinforcing neuronal lineage determination, to terminating neurogenesis. Findings from these studies indicate that we have to be careful about making conclusions regarding the neuronal potential of progenitors from the CNS, as oligodendrocyte precursor cells (glial progenitors that do not initially respond to neurogenic factors in culture) can acquire neuronal potential and CNS stem cell properties over time in culture [17••]. With regard to the response of neural stem cells to neurogenic factors, it has been demonstrated that neural stem cells undergo changes over time in vivo in their neurogenic properties while remaining multipotent and self-renewing. Cell-intrinsic changes in responsiveness to neurogenic signals, such as BMPs, influence the number and type of neurons that stem cells can make in vivo [18•,19•]. To reinforce neurogenesis, proneural basic helix–loop–helix (bHLH) transcription factors not only drive neurogenesis by activating the expression of a cascade of neuronal genes, but they inhibit the expression of glial genes [20,21•,22••]. Finally, if proneural genes inhibit gliogenesis, how is neurogenesis terminated so that gliogenesis can begin? Part of the answer is that Notch activation acts in neural stem cells as a switch that terminates neurogenesis and initiates gliogenesis, even in the continued presence of neurogenic growth factors [23•,24•,25••,26•,27•]. Each of these recent studies on different aspects of neural stem cell differentiation has implications for our understanding of important principles of the regulation of neurogenesis.
The neuronal potential of neural progenitors In the neural stem cell literature, the results from studies using cultured progenitors are strikingly inconsistent with
Neuronal potential and lineage determination by neural stem cells Morrison
those from studies using uncultured progenitors. Studies of cultured cells have emphasized the remarkable ability of stem cells from one region of the nervous system to give rise to neurons found in other regions [28–31]. This led to the idea of a common CNS stem cell with the potential to give rise to all types of neurons in the CNS. In contrast, studies of uncultured progenitors transplanted to a different region of the nervous system or to an earlier time in development have consistently observed restrictions in the ability of these stem cells to form the neurons that would normally form at these other places or times [32–35]. It seems unlikely that the higher purity of multipotent progenitors in the cultured CNS cell preparations (cultures favor the proliferation and survival of multipotent progenitors) were responsible for this difference. This is because, even when NCSCs are prospectively identified and purified by flow-cytometry from uncultured tissue, transplantation of these uncultured NCSCs in vivo reveals restrictions or biases in neuronal potential [19•]. Instead, it seems likely that neural progenitors may acquire a broader developmental potential as a result of proliferation in culture. The evidence that developmental potential can sometimes broaden in culture is presented in detail in an excellent recent review [36]. The most direct evidence that CNS progenitors can acquire a broader developmental potential comes from studies of oligodendrocyte precursor cells. After decades of studies in vitro and in vivo, these cells were thought to be glial committed, but recently were shown to acquire the ability not only to make neurons but to form neurospheres (CNS stem cell colonies) after being cultured in a series of growth factors [17••]. Indeed, it is possible to culture neurospheres from any region of the adult CNS, including regions that show no evidence of neurogenic activity in vivo [7,37]. One possible interpretation of this is that glial progenitors that lack neurogenic potential or multipotency in vivo may acquire the ability to make neurons and stem cell colonies in culture [38,39]. It will be important to determine whether the expression of patterning genes that specify region-specific neuronal fates in vivo is lost in culture and whether the loss of expression of such patterning genes leads progenitors to acquire a broader developmental potential in culture than they would have in vivo. To resolve these issues it will be necessary to continue to prospectively identify and purify neural stem cells [6,40,41] so that their properties can be studied in vivo as well as in vitro.
Changes in growth factor sensitivity regulate neuronal lineage determination How does a neural stem cell ensure that it produces the right number and kind of neurons? Do changes in the expression of neurogenic signals in the environment modulate the number and type of neurons produced? Or do cell-intrinsic changes in neural stem cells affect the way stem cells respond to neurogenic factors in the environment? Both types of mechanisms are likely to be involved, but work published over the past year has emphasized that
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neural stem cells change their cell-intrinsic properties over time in a way that influences the number and type of neurons they can generate. Hematopoietic stem cells [42–44], retinal stem cells [45], and cortical progenitors [46] have been demonstrated to undergo cell-intrinsic changes in developmental or neuronal subtype potential over time. Nonetheless, the literature on CNS stem cells has tended to emphasize the ways in which stem cells retain a broad neuronal potential rather than evidence of restrictions in neuronal potential. CNS stem cells undergo restrictions that reduce the number and types of neurons they make at later developmental stages. CNS stem cells from the postnatal subventricular zone failed to form large projection neurons or neurons in the cortex or hippocampus upon transplantation into the lateral ventricles of E15 fetuses [47]. This suggests that subventricular zone stem cells, which normally give rise to interneurons of the olfactory bulb, may have lost the potential to make projection neurons and certain other types of neurons that form in other regions of the CNS. A more recent paper by Temple and colleagues [18•] shows that the neurogenic properties of cortical stem cells undergo cell-intrinsic changes even over much shorter developmental times. Temple and colleagues [18•] isolated stem cells from the embryonic cortical ventricular zone and monitored the proliferation and differentiation of individual stem cells in culture. They found that the stem cells always made neurons first and glia second, just as they do in vivo. This suggested that the information required for the generation of neurons followed by glia was intrinsic to an isolated clone of stem cells and their progeny. Even more interesting was the observation that stem cells isolated from later embryonic stages gave rise to fewer neurons before initiating gliogenesis than stem cells isolated at earlier stages. This again mirrored what is observed in vivo. The molecular basis for this developmental change in neurogenic capacity was not determined but could relate to cell-intrinsic changes in sensitivity to lineage determination factors (reviewed in [48]). Within the PNS, analogous changes occur in NCSCs over time. Early migrating NCSCs (mNCSCs) can be isolated from E10.5 neural tube explants [4] while postmigratory NCSCs can be isolated from E14.5 sciatic nerves (snNCSCs) [6]. The neurogenic potentials of these two NCSC populations were compared by transplanting uncultured, purified rat NCSCs from either stage of development into the neural crest migration pathway of chick embryos [19•,49]. In this assay the rat NCSCs migrated along with chick neural crest cells and differentiated into diverse derivatives throughout the chick PNS. Both the mNCSCs and the snNCSCs gave rise to cholinergic neurons in parasympathetic ganglia, noradrenergic neurons in sympathetic ganglia, and glia in many locations, as would be expected; however, two differences were observed [19•]. The snNCSCs consistently gave rise to fewer neurons than the mNCSCs, and the snNCSCs only rarely gave rise to noradrenergic neurons in vivo, despite the fact that they
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readily gave rise to noradrenergic neurons in culture [50]. The snNCSCs also required 50-fold higher levels of BMP stimulation than the mNCSCs to initiate neurogenesis. As BMPs drive autonomic neurogenesis and higher levels of BMP stimulation were required for noradrenergic differentiation [19•], the data suggest that the decreased sensitivity of the snNCSCs led to a quantitative reduction in neurogenic capacity and a bias against noradrenergic differentiation in the presence of limiting BMP levels in vivo. Thus, just as with cortical CNS stem cells, NCSCs exhibit reduced neurogenic capacity with increasing developmental time and biases that reduce their ability to produce certain types of neurons, all while remaining multipotent and self-renewing. Changes in growth factor sensitivity over developmental time may be a general mechanism by which stem cells modulate their developmental potential and differentiation. It will also be interesting to determine whether changes in the sensitivity to neurogenic factors are behind the increased neurogenic potential of CNS progenitors in culture [17••,39].
Proneural bHLH genes inhibit gliogenesis Neurogenic factors promote neurogenesis by inducing the expression of proneural bHLH transcription factors such as Neurogenin and Mash-1. These proneural bHLH proteins are master regulators of neuronal differentiation that coordinate the expression of neuronal genes. For example, BMPs promote autonomic neurogenesis in the PNS by inducing the expression of Mash-1 in NCSCs [13]. Mash-1 then turns on a cascade of genes that collectively confer
Proneural genes such as Neurogenin1 promote neurogenesis by forming complexes with the CBP–p300–Smad1 transcriptional coactivator, binding to the promoters of neuronal genes, and activating transcription. Activation of the LIF receptor by CNTF promotes gliogenesis by activating STAT1/3. Activated STAT1/3 can also form a complex with the CBP–p300–Smad1 transcriptional coactivator, and bind to the promoters of glial genes, activating their transcription. Neurogenin1, and perhaps other proneural genes, can inhibit glial differentiation by sequestering the CBP–p300–Smad1 transcriptional coactivator to neuronal promoters and away from glial promoters, as well as by inhibiting STAT activation [22••]. BMPs can promote either neuronal differentiation or glial differentiation by activating Smad1, which binds to CBP–p300. In some cells, BMPs might also promote neuronal differentiation by promoting expression of proneural bHLH genes. Notch promotes gliogenesis and inhibits neurogenesis [23•,24•,25••,26•,27•]. It may do this by inhibiting the expression of proneural bHLH transcription factors and perhaps by promoting the transcription of glial genes by other mechanisms.
both pan-neuronal and subtype-specific aspects of autonomic neuronal identity [51]. Other types of proneural bHLH genes, like Neurogenin1 [52], promote differentiation into other types of neurons in the PNS and CNS. Three groups recently published evidence indicating that, in addition to acting as master regulators of neurogenesis, proneural bHLH transcription factors also inhibit gliogenesis [20,21•,22••]. Deficiencies in various combinations of proneural genes led not only to a loss of certain types of neurons, but to premature gliogenesis as well [20,21•]. One mechanism by which proneural genes might inhibit gliogenesis is by the sequestration of transcriptional coactivators away from glial promoters, preventing the expression of glial genes. Sun, Greenberg and colleagues [22••] presented evidence that Neurogenin1 can bind the CBP–Smad1 (CREB binding protein) transcription complex, preventing it from being recruited to glial promoters (Figure 1). By sequestering the CBP–Smad1 complex at neuronal promoters, proneural bHLH genes may both promote the expression of neuronal genes and inhibit the expression of glial genes. These observations resonate with the earlier discovery that transcription factors that regulate myeloerythroid differentiation in the hematopoietic system consistently act by promoting the acquisition of one fate while inhibiting the acquisition of alternative fates [53]. Thus, it may be a general strategy for lineagedetermining transcription factors to reinforce the process of differentiation by both promoting one fate and inhibiting alternative fates.
Neuronal potential and lineage determination by neural stem cells Morrison
Notch activation promotes gliogenesis Throughout the nervous system, neurogenesis occurs first and is followed by gliogenesis. How do neural stem cells terminate neurogenesis and initiate gliogenesis? Do the neurogenic signals just go away, or is neurogenesis actively shut down when gliogenesis begins? The expression of BMP2 or BMP4 near forming autonomic ganglia instructs NCSCs to differentiate into autonomic neurons [13,54–56]. But instead of turning off when it is time for gliogenesis to begin, BMP expression persists. So how does the second wave of neural crest progenitors undergo gliogenesis with the continued presence of the strongly neurogenic BMPs? An obvious possibility is a feedback signal from the neurons that would overcome the neurogenic influence of BMPs and promote gliogenesis. Indeed, the nascent neurons do express the gliogenic factor Nrg-1, but when NCSCs are simultaneously exposed to BMP2 and Nrg-1, neurogenesis continues and gliogenesis is completely suppressed [57]. Thus, Nrg-1 cannot explain the switch to gliogenesis. It was demonstrated recently that Notch activation in NCSCs instructs glial differentiation in a way that is dominant to the neurogenic influence of BMP2 and BMP4 [25••]. Furthermore, Notch activation caused glial lineage determination much more quickly than is observed in response to Nrg-1. Exposure to a soluble form of a Notch ligand (Delta) for only 24 hours caused an irreversible loss of neuronal potential, such that a subsequent 4 days of culture in BMPs resulted in continued glial differentiation, rather than neuronal differentiation. The transient expression of Notch ligands by nascent autonomic neurons may thus trigger a switch in NCSCs to terminate neuronal differentiation and initiate gliogenesis, despite the continued presence of BMPs. Is Notch activation a signal for the termination of neurogenesis and the initiation of gliogenesis throughout the nervous system? Notch pathway activation has also been observed to promote the acquisition of glial fates in vivo in the postnatal retina [23•] and in the fetal telencephalon [24•]. It was not clear in these cases whether Notch was instructing gliogenesis or just inhibiting neurogenesis, but in other studies of retinal neurogenesis in vivo, Notch pathway activation instructed the generation of Muller glia at the expense of neurons [26•,58]. Notch activation also instructs astrocytic differentiation by CNS stem cells from the adult hippocampus [27•]. Although there are several examples of cases in which Notch activation promoted gliogenesis by neural stem cells, there are also examples when Notch signaling was necessary or sufficient to maintain neural stem cells in an undifferentiated state [59,60]. Overall, it would appear that Notch signaling maintains multipotency in some neural stem cells but promotes glial differentiation in others or at other times during development. Another interpretation is that Notch activation may cause overt glial differentiation in some stem cells, whereas in other cases it may cause only glial lineage determination, with other factors additionally required for overt differentiation. One trend that may be emerging from the literature is that at early times of
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development, prior to the onset of gliogenesis, Notch activation may promote a maintenance of multipotentiality, but at later times during development, when gliogenesis has started or is about to start, it may promote gliogenesis. In this way, Notch activation would be modulated to have an effect that was appropriate to developmental stage. What is the molecular mechanism by which Notch promotes gliogenesis? As Notch inhibits the expression of Neurogenin, Mash-1, and other proneural bHLH genes [25••,52], it would be expected to promote gliogenesis by inhibiting the ability of proneural genes to inhibit gliogenesis (Figure 1; reviewed in [61]). Notch may also have other more direct mechanisms for promoting expression of glial genes. For example, Notch remained able to slightly promote expression of the glial marker GFAP (glial fibrillary acidic protein) even after mutation of the STAT3 (signal transducers and activators of transcription) binding site in the GFAP promoter [27•]. As the STAT3 binding site is where the STAT3–CBP–Smad1 complex activates transcription, this implies that part of the mechanism by which Notch promotes gliogenesis is independent of the CBP–Smad1 complex and, therefore, independent of the ability of proneural genes to sequester this complex. Notch probably functions in many regions of the nervous system as a switch that terminates neurogenesis and causes glial lineage determination, in part by inhibiting expression of proneural bHLH factors.
Conclusions Our understanding of neuronal lineage determination has advanced during the past year with the demonstration that proneural genes inhibit gliogenesis [20,21•,22••], and that Notch activation instructs gliogenesis [25••,27•]. We have also learned that neuronal potential is a moving target. Stem cells can reduce their sensitivity to neurogenic factors over time in vivo, reducing their capacity to form neurons and biasing the types of neurons they can make [18•,19•]. Progenitors that lack the ability to respond to neurogenic signals can acquire neurogenic potential in culture [17••]. We must stop thinking of neurogenic capacity and neuronal potential in black and white terms. The neuronal potentials of progenitors in vivo exist in shades of gray, shades that can change over time in vivo, and shades that can change (perhaps unphysiologically) in culture.
Acknowledgements I am supported as an Assistant Investigator of the Howard Hughes Medical Institute and a Searle Scholar. I apologize to those authors whose work was not cited due to space limitations. I thank Nancy Joseph and Theodora Ross for helpful comments on the manuscript.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
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10. Palmer TD, Takahashi J, Gage FH: The adult rat hippocampus contains primordial neural stem cells. Mol Cell Neurosci 1997, 8:389-404. 11. Horner PJ, Power AE, Kempermann G, Kuhn HG, Palmer TD, Winkler J, Thal LJ, Gage FH: Proliferation and differentiation of progenitor cells throughout the intact adult rat spinal cord. J Neurosci 2000, 20:2218-2228. 12. Panicker MM, Rao M: Stem Cell Biology. Edited by Marshak DR, Gardner RL, Gottlieb D. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 2001:399-438. 13. Shah NM, Groves A, Anderson DJ: Alternative neural crest cell fates β superfamily members. Cell are instructively promoted by TGFβ 1996, 85:331-343. 14. Gross RE, Mehler MF, Mabie PC, Zang Z, Santschi L, Kessler JA: Bone morphogenetic proteins promote astroglial lineage commitment by mammalian subventricular zone progenitor cells. Neuron 1996, 17:595-606. 15. Johe KK, Hazel TG, Muller T, Dugich-Djordjevic MM, McKay RDG: Single factors direct the differentiation of stem cells from the fetal and adult central nervous system. Genes Dev 1996, 10:3129-3140. 16. Li W, Cogswell CA, LoTurco JJ: Neuronal differentiation of precursors in the neocortical ventricular zone is triggered by BMP. J Neurosci 1998, 18:8853-8862. 17. ••
Kondo T, Raff M: Oligodendrocyte precursor cells reprogrammed to become multipotential CNS stem cells. Science 2000, 289:1754-1757. Oligodendrocyte precursor cells were studied for many years by many laboratories in vitro and in vivo and were thought to be committed to glial fates. Prospectively identified and purified oligodendrocyte precursor cells were not initially able to respond to neurogenic signals in culture, but after exposure to BMP-containing medium followed by FGF-containing medium, these cells not only made neurons, but they formed neurospheres and exhibited the properties of neural stem cells. These data strongly suggest that CNS cells that are committed to glial fates in vivo can reprogram their developmental potential in culture. The ability of oligodendrocyte precursors to adopt the properties of CNS stem cells over time in culture emphasizes the need to prospectively identify CNS stem cells to prove that cells exist in vivo with properties that are similar to the self-renewing, multipotent cells that have been characterized in vitro. 18. Qian X, Shen Q, Goderie SK, He W, Capela A, Davis AA, Temple S: • Timing of CNS cell generation: a programmed sequence of neuron and glial cell production from isolated murine cortical stem cells. Neuron 2000, 28:69-80. A program that brings about neuronal differentiation before glial differentiation is encoded intrinsically within CNS stem cells. Individual cells from the E10 cortex were cultured at clonal density and then the proliferation and differentiation of individual clones were followed by time-lapse video microscopy. In doing so, Qian et al. retrospectively constructed a ‘family tree’ for each stem cell clone. Multipotent progenitors in culture always generated neuroblasts before glioblasts; furthermore, multipotent progenitors from the E10 cortex gave rise to many more neurons than did multipotent progenitors from the E16 cortex even though there was no obvious difference in their capacity to generate glia. Thus, in addition to encoding the order of neurogenesis/ gliogenesis, stem cells also change over time in their propensity to generate neurons, despite remaining multipotent.
19. White PM, Morrison SJ, Orimoto K, Kubu CJ, Verdi JM, Anderson DJ: • Neural crest stem cells undergo cell-intrinsic developmental changes in sensitivity to instructive differentiation signals. Neuron 2001, 29:57-71. Migrating mNCSCs were purified from E10.5 neural tube explants while postmigratory snNCSCs were purified by flow-cytometry from E14.5 sciatic nerves [6]. When these cells were injected into the neural crest migration pathways of chick embryos the rat NCSCs engrafted throughout the chick PNS. Both mNCSCs and snNCSCs were nearly pure populations of self-renewing multipotent progenitors that gave rise to large numbers of cholinergic and noradrenergic neurons in vitro. But in vivo the snNCSCs consistently gave rise to 10-fold fewer neurons, and rarely gave rise to noradrenergic neurons. The snNCSCs were 50-fold less sensitive to the neurogenic activity of BMPs and noradrenergic differentiation required higher levels of BMP stimulation. This suggested that levels of BMPs were limiting in vivo but not in vitro for neuronal differentiation and the acquisition of noradrenergic identity. Cell-intrinsic changes in growth factor sensitivity in stem cells in vivo lead to quantitative reductions in the capacity for neuronal differentiation and biases in neuronal subtype potential, even while the stem cells remain multipotent and self-renewing. To detect mechanisms like this that regulate stem cell differentiation, neural stem cells must be prospectively identified [6] and studied in vivo. 20. Tomita K, Moriyoshi K, Nakanishi S, Guillemot F, Kageyama R: Mammalian achaete-scute and atonal homologs regulate neuronal versus glial fate determination in the central nervous system. EMBO J 2000, 19:5460-5472. 21. Nieto M, Schuurmans C, Britz O, Guillemot F: Neural bHLH genes • control the neuronal versus glial fate decision in cortical progenitors. Neuron 2001, 29:401-413. Nieto et al. analyzed mice that were deficient for Mash1 and/or Neurogenin2 and concluded that, in addition to reduced neurogenesis, glial precursors were generated earlier and in greater numbers in the double mutant mice. However, only a minority of animals exhibited premature GFAP expression, suggesting that loss of Mash1 and Neurogenin2 may accelerate glial lineage determination but that other factors determine the timing of overt differentiation. The appearance of an increase in the number of immature glia in double mutants in vivo, and the increased glial lineage determination observed in one subset of progenitors from Mash-1 deficient mice, suggest that proneural genes are necessary to avoid premature glial lineage determination, but loss of proneural genes is not sufficient to cause glial commitment. By comparing the results of Tomita et al. [20] to the results of Nieto et al., it appears that gliogenesis is accelerated to different extents in different regions of the nervous system, depending on which proneural genes are deleted. 22. Sun Y, Nadal-Vicens M, Misono S, Lin MZ, Zubiaga A, Hua X, Fan G, •• Greenberg ME: Neurogenin promotes neurogenesis and inhibits glial differentiation by independent mechanisms. Cell 2001, 104:365-376. Sun et al. observed that Neurogenin1 strongly inhibited LIF-induced (leukemia inhibitory factor) glial differentiation by cortical progenitors in culture, independent of its ability to promote neuronal differentiation. Within the GFAP promoter, the STAT binding site was necessary for the full Neurogenin1-mediated inhibition of GFAP expression but Neurogenin1 binding sites were not. The STAT binding site promotes GFAP transcription by binding a complex of STAT1/3 (activated by LIF/CNTF [ciliary neurotrophic factor] signaling), CBP or p300 (ubiquitously expressed transcriptional coactivators), and Smad1 (activated by BMP signaling). Neurogenin1 inhibits the assembly of this complex by binding to both CBP and Smad1, interfering with the ability of CBP–Smad1 to bind STAT3 (Figure 1). Thus, Neurogenin1 may inhibit glial lineage determination by sequestering the CBP–p300–Smad1 transcriptional complex away from the promoters of glial genes. 23. Furukawa T, Mukherjee S, Bao ZZ, Morrow EM, Cepko CL: rax, • Hes1, and notch1 promote the formation of muller glia by postnatal retinal progenitor cells. Neuron 2000, 26:383-394. Constitutively active Notch1 and Hes1, a downstream mediator of the transcriptional effects of Notch signaling, were overexpressed in the retinas of P0 rats in vivo. Overexpression of either gene caused an increase in the expression of Muller glial markers. However, it was unclear in these experiments whether Notch signaling was instructing glial differentiation, or whether glial differentiation occurred by default in response to an inhibition of neuronal differentiation. 24. Gaiano N, Nye JS, Fishell G: Radial glial identity is promoted by • Notch1 signaling in the murine forebrain. Neuron 2000, 26:395-404. Cells in the mouse fetal forebrain (telencephalon) were infected with a retroviral vector bearing the constitutively active Notch1 intracellular domain. Cells that expressed constitutively active Notch acquired a radial glial identity and then became periventricular astrocytes postnatally. As many radial glia and periventricular astrocytes are thought to be multipotent, it is not clear in these experiments whether Notch is promoting glial differentiation or a maintenance of multipotentiality.
Neuronal potential and lineage determination by neural stem cells Morrison
25. Morrison SJ, Perez S, Verdi JM, Hicks C, Weinmaster G, Anderson DJ: •• Transient Notch activation initiates an irreversible switch from neurogenesis to gliogenesis by neural crest stem cells. Cell 2000, 101:499-510. Most models of Notch function in vertebrate progenitors hold that Notch acts reversibly to inhibit differentiation, maintaining progenitors in an undifferentiated state. In this study, Notch activation not only instructed glial differentiation but it caused an irreversible loss of neuronal potential in NCSCs. For example, NCSCs were exposed to Notch ligand for only 24 hours, then the Notch ligand was washed out of the culture medium and replaced with medium containing the neurogenic factor BMP2. Instead of undergoing neuronal differentiation, the NCSCs continued to undergo glial differentiation despite the loss of Notch ligand and the presence of BMP2. These observations suggest that Notch activation in snNCSCs sends a positive signal that promotes gliogenesis rather than just a reversible negative signal inhibiting neurogenesis. 26. Hojo M, Ohtsuka T, Hashimoto N, Gradwohl G, Guillemot F, • Kageyama R: Glial cell fate specification modulated by the bHLH gene Hes5 in mouse retina. Development 2000, 127:2515-2522. Hes5, a downstream mediator of the transcriptional effects of Notch signaling, was necessary and sufficient to increase the numbers of Muller glia in the E15-P1 mouse retina. Because expression of Hes5 did not promote proliferation or cell death, and increased the numbers of glia while decreasing the number of neurons, it appeared that Hes5 instructed glial differentiation. Kageyama and colleagues, studies of retinal development suggest that while Hes5 promotes glial differentiation, Hes1 may promote a maintenance of multipotentiality. This suggests that there may be a bifurcation within the Notch pathway as different downstream mediators of Notch signaling may have different effects on progenitor differentiation. 27. •
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