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Cell, Vol. 96, 211–224, January 22, 1999, Copyright 1999 by Cell Press Progression from Extrinsic to Intrinsic Signaling in Cell Fate Specification: A View from the Nervous System Thomas Edlund*‡ and Thomas M. Jessell†‡ * Department of Microbiology University of Umea S-901 87 Umea Sweden † Howard Hughes Medical Institute Department of Biochemistry and Molecular Biophysics Columbia University New York, New York 10032 Introduction The diversity inherent in biological systems has its roots in genetic variation but is revealed through distinctions in the molecular profile and thus the identity of individual cells. The diversification of cell types is evident in an extreme form in vertebrate tissues, and amongst these, the nervous system contains perhaps the richest array of cell types. Even now, the number of distinct neuronal classes that exists is unclear, but traditional estimates of a few hundred mammalian neuronal subtypes appear to be overly conservative (Stevens, 1998). Attempts to understand the mechanisms that generate cell diversity through an analysis of vertebrate neural systems may therefore appear ill advised. Nevertheless, the generation of diverse neural cell types underlies in large part the remarkable information processing capacity of the central nervous system. Thus, one goal of studies of neural cell fate determination not attainable through the use of other tissues is to understand the logic that controls the later assembly of neuronal circuits. Problems posed by the number and complexity of neuronal subtypes may be partly offset by the fact that the mechanisms used to establish neural cell diversity in vertebrates are in many cases conserved with those in other tissues and more primitive organisms. The central issue in the specification of cell fate is the interaction between two general sets of determinative factors: secreted or transmembrane (extrinsic) signals present in a cell’s local environment and intrinsic signals that operate in a cell-autonomous manner. Cell identities are assigned through the interplay of both sets of factors, but the relative contribution of each set varies with cell type and developmental time. The task then, is to define how these various environmental, intrinsic, and temporal controls cooperate in establishing the identity of individual cells. Secreted and cell surface proteins control cell fates in a variety of different ways (see Lawrence and Struhl, 1996; Gurdon et al., 1998). These strategies are summarized here only briefly (Figure 1; Table 1), and in this article we instead focus on the issue of how vertebrate neural cells gradually acquire independence from extrinsic signals and become progressively more reliant on intrinsic programs of differentiation (Figure 2A). We examine when this transition occurs and the potential ‡ E-mail: T. E., [email protected]; T. M. J., tmj1@ columbia.edu. Review mechanisms through which it may be achieved. Neuronal differentiation represents an extreme version of such a transition, since it appears to be accompanied by the loss of potential for reentry into the cell cycle and for dedifferentiation. We therefore also address the contribution of cell cycle exit to the acquisition of neural cell identity. Since many of the principles of cell fate specification are highly conserved, we have in some instances also incorporated insights derived from the analysis of nonneural systems and invertebrate organisms. Many other related issues, in particular the nature and properties of stem cells (Morrison et al., 1997; Gage, 1998; Panchision et al., 1998), the lineage relationships of neural cells (Cepko et al., 1998), the mechanisms of asymmetric cell division (Lu et al., 1998), and developmental cell death (Pettemann and Henderson, 1998) have recently been discussed elsewhere and are not addressed in detail. The Timing of Restrictions in Neural Cell Fate The differentiation of multipotential neural progenitor cells into specific classes of postmitotic neurons or glial cells occurs over a protracted period and appears to be accompanied by a progressive restriction in the range of fates available to individual cells. The time at which such restrictions occur has generally been examined either by placing progenitor cells from different developmental stages in ectopic environments in vivo or by exposing cells to defined signals in vitro and monitoring changes in response and fate. These manipulations have revealed that restrictions in cell fate occur at many different stages and times. The fate of certain classes of neural cells appears to be restricted several divisions before cell cycle exit, but for other cell types, similar restrictions appear to occur much closer to the time of cell cycle exit, and for yet others, only after they have achieved a postmitotic state. In the following sections, we review briefly some of the evidence that has led to these general conclusions. Restrictions in Developmental Potential Prior to Cell Cycle Exit The neural crest represents the cell type that has provided the most detailed information on the mechanisms that control the fate of multipotential neural progenitors. The suggestion that progressive restrictions occur in the developmental potential of neural crest cells first emerged from studies on transplanted or cultured neural crest cells (Le Douarin, 1986; Artinger and Bronner-Fraser, 1992; Dupin et al., 1998). However, early transplantation studies were limited to the analysis of populations of cells, and many in vitro studies have monitored cell fate decisions in the absence of manipulation of the local environment. As a consequence, clear evidence for the existence of subtype-restricted neural crest progenitor cells has been surprisingly difficult to obtain (Anderson, 1989). More recent studies have, however, begun to indicate that certain neural crest–derived progenitor cells are indeed committed to a specific fate several cell divisions prior to their exit from the cell cycle. Cell 212 Figure 1. Strategies of Extrinsic Signaling in Cell Fate Specification (A) Inductive signaling. Adjacent cells acquire different fates through the selective exposure of one cell to a locally acting extrinsic signal. (B) Gradient signaling. Extrinsic signals are capable of directing distinct cell fates at different concentration thresholds. Studies of cell patterning in Drosophila have provided evidence that Wingless (Wg) and Decapentaplegic (Dpp) can generate distinct cell types through actions on target cells at different concentration thresholds (Lawrence and Struhl, 1996). In Xenopus embryos, discrete changes in mesodermal gene expression and cell fate are elicited by 2- to 3-fold differences in the concentration of activin (Gurdon et al., 1998). The graded signaling activity of Sonic hedgehog (Shh) appears to be required for the generation of neural cell types in the ventral neural tube (Ericson et al., 1997). (C) Antagonist signaling. Many secreted factors (blue) that control cell fate are themselves targets of secreted inhibitory factors (red) (Table 1). One hallmark of such inhibitory factors is that they do not interact with specific cell surface receptors but instead bind directly to signaling ligands and/or their receptors, blocking their signaling function (Schweitzer et al., 1995; Thomsen, 1997; Zorn, 1997; Hacohen et al., 1998; Hsu et al., 1998). Neural induction in vertebrates may be mediated in part through such a mechanism (Wilson and Hemmati-Brivanlou, 1997). The existence of dedicated antagonists for certain secreted factors can also result in a negative feedback control of the net level of signaling and result in further distinctions in cell pattern as, for example, in the patterning of the Drosophila oocyte (Wasserman and Freeman, 1998). (D) Cascade signaling. Some secreted proteins achieve long-range patterning through the initiation of cascades of diffusible factors (see Lawrence and Struhl, 1996). (E) Combinatorial signaling. Cells may acquire distinct identities through their coincident exposure to two different signals, the activities of both cooperating to generate a cell type that is not obtained by the action of either signal in isolation. Direct evidence for combinatorial signaling of this type has been hard to obtain, since it requires the demonstration that signals act in a coincident rather than sequential manner. One such interaction may involve Dpp and Wg signaling and direct cell growth and pattern along the proximodistal axis of appendages in Drosophila (Lecuit and Cohen, 1997; Campbell and Tomlinson, 1998). In vertebrates, the conjunction of BMP and Sonic hedgehog signaling may specify the identity of a set of ventral midline diencephalic cells (Dale et al., 1997). (F) Lateral signaling. A distinct mechanism for establishing cell identity uses a signaling system in which small differences in the level of signals transmitted between interacting cells are rapidly amplified by a feedback mechanism to generate marked differences in the level of intracellular signals that subsequently direct distinct fates. This mode of signaling operates in many tissue types and in widely divergent organisms and is typically mediated by the activation of the notch class of receptors (Simpson, 1997). Local sources of extrinsic signals can modify the state of notch signaling and thus bias cell fate decisions. The fringe protein appears to modulate notch signaling in this way (Panin et al., 1997). In all diagrams, I indicates the cell that provides the source of an inductive signal, A, B, and C indicate distinct cell fates, and gray color indicates an unspecified cell. One analysis has focused on a population of neural crest–derived progenitor cells present in the embryonic gut primordium. These cells can be identified by expression of c-Ret, a subunit of the receptor for the glialderived neurotrophic factor (GDNF) family. Isolated c-Ret1 progenitor cells divide multiple times in vitro, yet they eventually differentiate in a synchronous manner and give rise exclusively to neurons (Lo and Anderson, 1995). Moreover, neuronal fate is preserved even when cells are challenged in vitro with extrinsic signals such Table 1. Conserved Classes of Cell Surface and Secreted Factors with Inductive Activities Vertebrates Drosophila C. elegans Secreted Inhibitor EGF/TGFa/neuregulin TGFb/BMP/activin spitz, vein dpp, 60A, screw lin-3 daf-7, unc129 FGF wnt delta, serrate hedgehog fringe branchless wg, d-wnts delta, serrate hedgehog fringe egl-17 mom-2, lin-44 lag-2, apx-1 — — argos noggin, chordin/sog follistatin, DAN, gremlin, cerberus sprouty frzB class — — — A selected list of classes of vertebrate proteins with inductive signaling activities that possess counterparts in Drosophila and/or C. elegans. Secreted factors that bind to and inhibit the actions of many of these proteins have also been identified. For details, see legend to Figure 1 and text. Review 213 Figure 3. Specification of Neuronal Fate Late in the Final Progenitor Division Cycle Figure 2. A Molecular Basis for the Regulation of Neural Competence to Extrinsic Signals (A) Neural crest–derived progenitor cells isolated from the enteric nervous system express the basic HLH protein Mash1. Maintained expression of Mash1 initially requires BMP signaling, but cells gradually acquire the ability to generate autonomic neurons independent of further BMP exposure. (B) Mash1 expression is required for maintained competence to respond to the neurogenic actions of BMPs. Top: After early removal of BMP signaling, Mash1 expression is rapidly extinguished and cells can no longer respond to the later neurogenic actions of BMPs. Middle: Introduction of Mash1 into neural crest–derived cells is not sufficient to promote neuronal differentiation. Bottom: Introduction of Mash1 into neural crest–derived cells maintains the competence of cells to respond later to the neurogenic actions of BMPs. N signifies neuronal fate. Adapted from Lo et al. (1997). as glial growth factor that are potent suppressors of neuronal differentiation in earlier stage neural crest cells. These results support the idea that some neural crest cells that emerge from the neural tube are initially multipotent but give rise to lineage-restricted progenitors that retain a limited capacity for cell division. Studies of mammalian cortical development have also provided evidence that CNS progenitor cells grown in vitro can retain the memory of extrinsic signals that promote region-specific phenotypic properties through one or more cell divisions after removal from such signals (Eagleson et al., 1997; Lillien, 1998). Together these findings are consistent with the idea that the fate of certain progenitor cells in both the peripheral and central nervous systems can be restricted before their final cell division cycle. Restrictions in Developmental Potential Late in the Final Progenitor Cell Cycle For certain cell types, it nevertheless appears that critical aspects of neural phenotype are acquired only late in the final division cycle of the progenitor cell. In the developing mammalian cerebral cortex, for example, (A) Cortical neuron specification. Progenitor cells present at early developmental stages in the ventricular zone of the cerebral cortex are fated to generate deep layer neurons. Transplantation of young progenitors into the ventricular zone of older host animals shows that these cells become restricted to a deep layer fate only late in their final progenitor division cycle. Cells that are transplanted in G1 or early S phase or that undergo further rounds of cell division in the older host environment change their properties and generate upper layer neurons. The transition of progenitor cells from a gray to brown state indicates an apparent restriction in area-specific fate (see Eagleson et al., 1997). Other aspects of the diagram adapted from data of McConnell and Kaznowski (1991). (B) Motor neuron specification. Exposure of Pax31, Pax61, Pax71 neural progenitor cells to Sonic Hedgehog (Shh) generates Pax61 ventral progenitors. These cells retain a dependence on Shh signaling for several rounds of cell division, acquiring independence late in their final progenitor division, at the time that they express the homeodomain proteins MNR2 and Lim3. Postmitotic motor neurons extinguish Pax6 and MNR2 expression and begin to express the homeodomain proteins Isl1, Isl2, and HB9. Adapted from data of Ericson et al. (1996) and Tanabe et al. (1998). the laminar fate of progenitor cells is acquired during the final progenitor cell division. This was revealed by the transplantation of young ventricular zone progenitors destined to generate deep layer neurons into an older host environment that is populated by progenitor cells fated to form upper layer neurons (McConnell and Kaznowski, 1991; Bohner et al., 1997). The grafted young progenitor cells become restricted to their deep layer neuronal fate only late in their final cell division cycle (Figure 3A). In contrast, cells grafted at earlier stages in the cell cycle or that undergo further rounds of cell division act as their older counterparts, migrating to upper layers of the cortex. The nature of the environmental signals that control the laminar fate of cortical neurons remains to be identified. In the developing spinal cord, a defined extrinsic signal, Sonic hedgehog (Shh), is thought to direct the fates of many neuronal types, including motor neurons and ventral interneurons (Ericson et al., 1997). The time at which progenitor cells fated to become motor neurons Cell 214 attain independence from Shh signaling has been examined in vitro by challenging neural cells with Shhblocking antibodies (Ericson et al., 1996) (Figure 3B). The generation of motor neuron progenitors requires an early phase of Shh signaling, but induced progenitors remain dependent on Shh signaling for several further rounds of cell division. These cells progress to a state of independence from Shh only late in their final progenitor cell cycle (Ericson et al., 1996) and at this point appear to commit to a motor neuron fate (Tanabe et al., 1998). These observations therefore provide evidence that certain neuronal progenitor cells in the CNS change their sensitivity to extrinsic signals, and thus their developmental potential, late in their final cell division cycle. A Basis for Temporal Changes in Progenitor Cell Response to Extrinsic Signals Observations of the type described above imply that neural progenitor cells gradually change their sensitivity to specific extrinsic signals. In addition, neural progenitor cells can maintain their sensitivity to a given signal over time but produce distinct cell types at sequential developmental stages (Michelsohn and Anderson, 1992; Liem et al., 1997). Generally, the basis of such temporal changes in progenitor cell competence to extrinsic signaling is not well understood (Lillien, 1998). In some instances, however, the change in competence of cells to extrinsic signals appears to involve the developmental regulation of expression of surface membrane receptors. Studies of neural differentiation in the retina and cerebral cortex have shown that progenitor cells exhibit temporal changes in their response to mitogenic factors of the epidermal growth factor (EGF) family (Lillien and Cepko, 1992; Lillien, 1995; Lillien and Wancio, 1998). Progenitor cells isolated at early stages of retinal development express low levels of EGF receptors, whereas progenitor cells isolated from later stages express a higher EGF receptor number. The level of EGF receptor expression appears to be a determinant of cell fate, since the exposure of older progenitor cells to low concentrations of EGF ligands stimulates their proliferation, whereas higher concentrations suppress proliferation and promote the differentiation of glial cells at the expense of rod photoreceptors. Evidence that EGF receptor levels may normally be limiting in the selection of cell fate has come from experiments in which retrovirally driven expression of additional EGF receptors in late-stage retinal progenitor cells suppresses their proliferation and increases the generation of glial cells. However, in younger progenitors an elevation in the level of EGF receptor signaling is not sufficient to specify glial fate, indicating a requirement for additional temporally regulated factors. Developmental changes in the level of EGF receptor expression have also been detected on progenitor cells in the cerebral cortex (Burrows et al., 1997) and may similarly contribute to the timing of cortical progenitor cell maturation. An insight into the transcriptional basis of changes in progenitor cell competence has emerged from studies of the differentiation of neural crest cells into autonomic neurons. This program of neurogenesis involves the activity of a basic–helix-loop-helix (bHLH) transcription factor, Mash1 (Lo et al., 1997) (Figure 2). The expression of Mash1 in neural crest cells is induced by members of the bone morphogenetic protein (BMP) subclass of transforming growth factor b proteins. The maintained exposure of these cells to BMPs leads to neuronal differentiation. However, neural crest–derived cells that initially express Mash1 but are not exposed further to BMPs rapidly stop expressing Mash1 and, in parallel, lose their competence to respond to later BMP exposure with neuronal generation. Forced expression of Mash1 in these neural crest cells is not sufficient to promote neurogenesis but is able to maintain the competence of these cells to the subsequent neurogenic actions of BMPs (Lo et al., 1997). These findings implicate Mash1 as a key factor in the competence of neural crest cells for neurogenic differentiation. A dynamic profile of bHLH gene expression is evident in many other regions of the developing vertebrate nervous system (see Sommer et al., 1996; Henrique et al., 1997). bHLH proteins may therefore have a more general role in the temporal regulation of neural cell responses to extrinsic signals. Specification of Neural Cell Identity after Cell Cycle Exit Although restrictions in cell fate clearly occur in progenitor cells, several lines of evidence indicate that extrinsic signals can also impose certain phenotypic properties on postmitotic neural cells. A late target-dependent switch in one aspect of neuronal phenotype, neurotransmitter synthesis and release, occurs in developing autonomic neurons (Landis, 1990) (Figure 4A). A subset of sympathetic neurons that innervate selected target cells in the periphery, notably the sweat gland cells of the foot pad, undergoes a developmental switch in their neurotransmitter properties. These neurons lose their initial noradrenergic transmitter phenotype and subsequently acquire cholinergic properties (see Landis, 1990). This switch appears also to be controlled by ciliary neurotrophic factor (CNTF)-like proteins secreted by the glandular target cells (Habecker et al., 1997). Studies of motor and sensory neuron development have also suggested a requirement for peripheral axonal projections in the establishment of mature patterns of neuronal gene expression. Sets of motor and sensory neurons that later interconnect in functional circuits can be defined by expression of members of the ETS class of transcription factors (Ghosh and Kolodkin, 1998; Lin et al., 1998). The onset of ETS protein expression occurs well after motor and sensory neurons have left the cell cycle and after their axons have reached the periphery. Moreover, the initiation of expression of these genes appears to depend on the early exposure of axons to signals provided by the developing limb target. These data raise the possibility that the peripheral control of neuronal ETS gene expression mediates the influence of the limb on the formation of selective connections between muscle sensory afferents and motor neurons (Wenner and Frank, 1995). Together, these findings provide evidence that the assignment of selected features of neuronal subtype identity can be imposed by extrinsic signals after the exit of neural cells from the cell cycle. Analysis of the development of photoreceptors in the rodent retina has provided additional evidence that postmitotic neural cells can exhibit a prolonged period of sensitivity to extrinsic signals (Figure 4B). Rod photoreceptors leave the cell cycle many days before they express cell type–specific differentiation markers such as rhodopsin (Ezzeddine et al., 1997; Neophytou et al., Review 215 Figure 4. Acquisition of Neuronal Subtype Identity after Cell Cycle Exit (A) A late switch in the phenotype of sympathetic neurons. Neural crest cells leave the cell cycle and generate noradrenergic sympathetic neurons (SN). Neurons that innervate sweat gland targets (T) initially release the transmitter norepinephrine (NA) and trigger the secretion of LIF/CNTF–like factors from the target cell. Exposure of sympathetic noradrenergic neurons to LIF/CNTF results in a switch in their transmitter properties, and these neurons now acquire a cholinergic phenotype (Sc) and release acetycholine as transmitter. Adapted from data in Landis (1990) and Habecker and Landis (1994). (B) Control of retinal neuronal phenotype after cell cycle exit. Rod photoreceptors (R) differentiate from cells that have left the cell cycle many days earlier. Exposure of postmitotic incipient photoreceptors to LIF or CNTF, factors derived from Muller glial cells (M), prevents photoreceptor differentiation and may promote bipolar cell (B) fate. Adapted from data of Neophytou et al. (1997) and Ezzeddine et al. (1997). 1997; Morrow et al., 1998). The differentiation of incipient photoreceptors can be arrested during this postmitotic period by exposure to factors normally provided by Muller glial cells, notably members of the CNTF/leukemia inhibitory factor (LIF) family. Cells only become refractory to the inhibitory actions of CNTF close to the time of onset of rhodopsin expression. Morover, some prospective photoreceptors that have been exposed to CNTF may acquire phenotypic characteristics of bipolar cells, a distinct retinal neuronal subtype (Ezzeddine et al., 1997). This finding suggests the intriguing possibility that, in addition to a refinement of the phenotypic properties of neuronal subsets, a more complete switch in neuronal subtype fate may be attainable after cell cycle exit. Mechanisms of Progression from Extrinsic to Intrinsic Signaling How might neural cells acquire independence from extrinsic signals? In other cellular contexts, strategies that permit cells to establish autonomous programs of differentiation and function have been defined. Some of these mechanisms may also apply to the developmental transition of neural cells, and we consider briefly three mechanisms of potential relevance: the generation of persistently active forms of intracellular transduction proteins, transcriptional autoactivation, and the long-term stabilization of states of gene expression (Figure 5). Persistent Activation of Intracellular Transduction Proteins The relay of extrinsic signals from the cell surface to the nucleus typically is mediated by cytoplasmic proteins whose activities are subject to posttranslational regulation. This feature has been accompanied by the emergence of mechanisms to alter the state of activity of cells for long periods, despite the decay of the stimulus that initially elicited the switch in cell activity. Studies of the biochemical basis of memory storage in neurons in particular have provided a precedent for ways in which long-term changes in cell states can be achieved through posttranslational regulation (see Schwartz, 1993; Lisman, 1994). One mechanism involves proteolytic processing of intracellular effector proteins, notably protein kinases (Figure 5A) (see Schwartz, 1993; Chain et al., 1995). Studies on the developmental control of cell fate have revealed instances of the regulation of intracellular signal transduction pathways through proteolysis. For example, activation of notch signaling appears to require the ligand-dependent proteolytic cleavage of its intracellular domain (Schroeter et al., 1998; Struhl and Adachi, 1998). The dorsoventral patterning of the early Drosophila embryo involves activation of the rel/NFkB family transcription factor dorsal and requires the ligandinduced proteolysis of its inhibitory subunit, the cactus protein (Belvin et al., 1995). In principle, ligand-dependent proteolytic processing could have a role in generating activated forms of intracellular transduction proteins and, if these activated proteins are stable, result in a long-term change in the state of cell differentiation. The neuronal protein kinases that function as mediators of extrinsic signals implicated in memory storage are also subject to persistent activation by intramolecular or intermolecular phosphorylation (see Schwartz, 1993) (Figure 5A). The potential contribution of phosphorylation-dependent mechanisms of persistent protein kinase activation to developing neural systems has not been examined in detail. However, the activity of many transcription factors expressed in neural cells is dependent on their phosphorylation state (see SassoneCorsi, 1995; Fowles et al., 1998; Jacobs et al., 1998; Zhong et al., 1998 and references therein), providing a potential link between long-term changes in protein kinase activity and the transcriptional control of neural cell fate. Transcriptional Autoactivation One mechanism by which transcription factors, once activated, can maintain states of gene expression is through a positive feedback loop that involves transcriptional autoregulation (Figure 5B). Many examples of autoregulatory feedback have been described during cell fate specification in other vertebrate tissues, notably muscle cells (Molkentin and Olson, 1996), and in Drosophila and C. elegans tissues (see Schier and Gehring, 1992; Xue et al., 1993). Cell 216 Figure 5. Possible Mechanisms of Progression from Extrinsic to Intrinsic Signaling during Cell Fate Specification The diagram shows three mechanisms for the progression and stabilization of neural cell identity: (A) persistent activation of protein kinases, (B) transcriptional autoactivation, and the (C) late stabilization of gene expression. In (A), the top diagram indicates the generation of a stable catalytically active kinase (orange) by the proteolytic degradation of an inhibitory subunit. The lower diagram depicts a phosphorylation-dependent conversion of a catalytically inactive kinase (gray) into an active kinase (orange). The state of kinase activation is perpetuated by autophosphorylation (anticlockwise arrow). In (B), the transcription of a gene encoding one transcription factor (orange) is initiated by the actions of a distinct transcription factor (blue) that is induced or activated by extrinsic signals. The state of transcriptional activation is perpetuated by positive autoregulatory feedback (orange). In (C), initial states of transcription are maintained through the actions of activators such as the trithorax group genes (trxG) or repressors such as the Polycomb group genes (PcG). For details, see text. I, inducing cell. Transcriptional autoactivation may also contribute to the acquisition of autonomous programs of differentiation in vertebrate neural cells. This mechanism is evident in the allocation of regional cell fate along the anteroposterior axis of the developing hindbrain. Here, cell identity depends in part on the establishment of segmental domains of Hox gene expression that in turn are defined by auto- and cross-regulatory feedback interactions between Hox genes (Nonchev et al., 1997). As one example, the initial pattern of expression of Hoxb4 in the hindbrain depends on transient inductive signaling from the paraxial mesoderm and involves a retinoid signaling pathway that leads to the activation of a cisacting retinoic acid response element within the Hoxb4 gene (Gould et al., 1998). The later phase of Hoxb4 expression, however, is independent of retinoid signaling and is maintained by Hox protein feedback regulation. The cross-regulation of closely related transcription factors is also apparent for several other classes of homeodomain proteins (see Pattyn et al., 1997). Studies of transcriptional responses to Shh signaling in the ventral neural tube have indicated additional roles for autoactivation in the maintenance of cell identity. A high concentration of Shh induces the expression of a winged helix transcription factor, HNF3b, and the expression of this factor is sufficient to direct floor plate differentiation (Sasaki and Hogan, 1994; Ruiz i Altaba et al., 1995). The expression of HNF3b is autoactivated in neural tube cells (Sasaki and Hogan, 1994), providing a potential mechanism by which floor plate cells maintain an autonomous state of differentiation independent of further Shh signaling. Similarly, the induction of motor neuron progenitors in response to a lower concentration of Shh is associated with the expression of a homeodomain protein, MNR2, that is sufficient to direct neural progenitor cells to a motor neuron fate (Tanabe et al., 1998). MNR2 expression is initiated during the final division cycle of motor neuron progenitors at the time that they attain independence of Shh signaling, and the gene can autoactivate its own expression (Tanabe et al., 1998). MNR2 autoactivation may therefore contribute to the conversion of Shh-dependent ventral progenitors to a committed motor neuron progenitor state. In addition, MNR2 may limit the duration of this committed progenitor state, since cells leave the cell cycle soon after the onset of MNR2 expression. Neural cells may also maintain states of cell differentiation through the activation of expression of closely related transcription factors that possess equivalent activities: the transcriptional counterpart of homeogenetic (like begets like) induction. This strategy may have its basis in instances of gene duplication during vertebrate evolution (Sidow, 1996) that result in the spatial conservation of expression of closely related transcription factors (see for example Hanks et al., 1995). During the MNR2-mediated induction of motor neuron differentiation, one of the target genes activated in postmitotic motor neurons is a closely related and functionally equivalent homeobox gene, HB9 (Tanabe et al., 1998). The expression of HB9 is maintained for a prolonged period in postmitotic neurons, perhaps compensating for the eventual extinction of MNR2 expression. Thus, transcription factors with divergent regulatory elements but conserved functions may provide a means of maintaining transcriptional activities at sequential steps in the program of differentiation of a single neural cell. Long-Term Stabilization of Gene Expression The stable specification of cell identity in many developing tissues requires mechanisms that maintain patterns of gene expression over long periods of time and in some instances through multiple rounds of cell division. The maintenance of body segment identity in Drosophila, for example, depends on the sustained expression Review 217 of homeotic genes, a process that is mediated by cisregulatory regions that contain targets for the Polycomb group (PcG) and trithorax group (trxG) genes (Gerasimova and Corces, 1998; Paro et al., 1998). PcG proteins generally maintain inactive states of homeotic gene expression, whereas trxG proteins sustain active states (Figure 5C). Mutations in PcG genes lead to the loss of repression of homeotic genes and the misspecification of segment identity (Bienz and Muller, 1995). Vertebrate PcG homologs have been identified, and many of these appear to have related functions in maintaining states of repression of homeotic genes (Gould, 1997; Schumacher and Magnuson, 1997). Loss-of-function mutations in some of these mouse genes result in ectopic Hox gene expression and developmental defects that include transformations in cell identity along the anteroposterior axis of the early embryo (see Gould, 1997). Establishing and maintaining such patterns of gene expression is dependent on chromatin architecture (Felsenfeld et al., 1996; Tsukiyama and Wu, 1997). Studies of gene silencing in many different systems suggest that repressed chromatin states are transiently erased at each cell division and reestablished in daughter cells (see Cavalli and Paro, 1998). The disassembly of chromatin during cell division may therefore permit extrinsic signals to alter programs of gene expression efficiently in dividing progenitor cells. However, since neurons and glial cells retain their differentiated properties in the absence of cell division, it remains an open question whether the mechanisms that maintain states of gene expression through mitotic divisions operate in postmitotic neural cells. An additional unresolved issue is whether transcription factors that direct the fate of neural progenitor cells also directly influence chromatin organization. Some evidence that this might be the case has come from studies of bHLH protein function in skeletal muscle differentiation. MyoD and Myf5 can efficiently remodel chromatin at binding sites in muscle gene enhancers and can also activate transcription at previously silent loci (Gerber et al., 1997). The ability of MyoD to activate endogenous muscle genes depends not on the bHLH or activation domains but instead on a distinct cysteine/histidine–rich region as well as a region in the carboxyl terminus of the protein, both of which are also conserved in Myf5. These findings suggest that a subset of myogenic bHLH proteins can participate directly in chromatin reorganization. The erythroid transcription factor GATA1 has been shown to perturb nucleosome structure (Boyes et al., 1998) and thus may also contribute to the generation of transcriptionally active chromatin states. It is possible, therefore, that transcription factors that direct neuronal fate do so in part through their ability to participate directly in the reorganization of chromatin structure. Cell Cycle Exit and the Acquisition of Neural Fates The transition of proliferative progenitor cells into postmitotic neurons or glial cells frequently coincides with the onset of expression of genes that define their generic properties. These observations raise two issues: what intrinsic mechanisms regulate the decision of neural progenitor cells to leave the cell cycle, and what does cell cycle exit contribute to the specification of neural cell fate? Regulation of Cell Cycle Exit Progression through the cell division cycle is driven by the actions of cyclin-dependent kinases (CDKs) and their activating cyclin subunits. CDK activity is suppressed through interactions with two major classes of inhibitory proteins: the Ink4 class that exhibits selectivity for CDKs 4 and 6 and the Cip/Kip class that shows a broader spectrum of CDK inhibitory activity (Sherr and Roberts, 1995; Harper 1997). The exit of neural cells from the cell cycle appears to occur at a restriction point in the G1 phase of the cell cycle (Zetterberg et al., 1995), and as a consequence, cells enter a G0-like state and begin to disassemble components of the core cell cycle machinery. Progress in understanding the relationship between cell cycle exit and cell differentiation during development has derived from studies of many vertebrate and invertebrate cell types. Many extrinsic signals, including secreted growth factors with inductive activities (see Massague and Polyak, 1995; Horsfield et al., 1998 and references therein) and neurotrophic factors (ElShamy et al., 1998) regulate the expression and/or activity of proteins that direct the cell cycle. Here we focus on the developmental roles of intrinsic factors, and in particular on CDK inhibitors, while appreciating that many other components of the core cell cycle machinery are likely to influence cell cycle exit in developing neural cells (Bartek et al., 1997; Lehner and Lane, 1997; Mulligan and Jacks, 1998). In vertebrates, studies of skeletal myogenesis have provided an informative precedent for considering how the regulation of neural cell cycle exit may intersect with programs of cell fate determination (see Piette, 1997). The commitment of progenitor cells to a muscle fate depends on the activation of members of the MyoD family of bHLH proteins, notably MyoD itself and Myf5, and precedes the point of cell cycle arrest (Walsh and Perlman, 1997; Zabludoff et al., 1998). Nevertheless, the execution of an overt myogenic differentiation program depends on cell cycle exit and reflects an inhibition of the activity of MyoD and related bHLH proteins under conditions of cell proliferation and high CDK activity (Lassar et al., 1994; Skapek et al., 1995; see Walsh and Perlman, 1997). This constraint on MyoD function can be overcome by inactivation of CDKs through expression of Cip/Kip class CDK inhibitors such as p21Cip (p21) and p27Kip (p27). Moreover, MyoD expression is itself able to activate p21 transcription (Halevy et al., 1995), an action that may contribute to its myogenic function. Roles for Cip/Kip proteins in promoting cell cycle exit in developing cells have also emerged from studies of Drosophila development (de Nooij et al., 1996; Lane et al., 1996). The terminal division of many vertebrate neural cells is accompanied by dynamic changes in the level of expression of Cip/Kips (Van Lookeren et al., 1998; Watanabe et al., 1998), suggesting an involvement of CDK inhibitors both in the the exit of neural progenitor cells from the cell cycle and in maintenance of the postmitotic state. At present, little is known about the intrinsic factors that regulate CDK activity in differentiating neurons. However, in vertebrates, as in Drosophila, neurogenesis Cell 218 appears to involve the sequential activation of bHLH factors that contribute both to the determination of neuronal fate and later to the promotion of aspects of neuronal differentiation (Anderson and Jan 1997; Lee 1997). Moreover, in Xenopus embryos a subset of these bHLH proteins, neurogenins 1 and 2 and NeuroD, is sufficient to promote the ectopic expression in ectodermal cells of markers characteristic of postmitotic neurons (Lee et al., 1995; Ma et al., 1996; Olson et al., 1998). Thus, it seems possible that the actions of certain neurogenic bHLH factors will intersect with the core cell cycle machinery, perhaps as with MyoD, through the induction of expression or activation of CDK inhibitors. The most detailed information on the regulation and role of Cip/Kip proteins in vertebrate neural cells has, however, emerged from studies of oligodendrocyte differentiation (Raff et al., 1998) (Figure 6). Maintained proliferation of oligodendrocyte progenitors normally depends on the presence of mitogenic factors such as PDGF. But even in the presence of saturating levels of mitogen, these progenitors will eventually exit the cell cycle and, in the presence of appropriate hormones, differentiate into oligodendrocytes (Figure 6A). Moreover, in vitro, the clonal progeny of an individual oligodendrocyte progenitor cell differentiate in near synchrony, suggesting the existence of a cell-intrinsic mechanism that controls the timing of cell cycle exit (Temple and Raff, 1986). The possibility that p27 might be a component of this cell-intrinsic timing machinery was suggested by studies showing that p27 protein levels accumulate with time in oligodendrocyte progenitor cells and increase further upon oligodendrocyte differentiation (Durand et al., 1997; Tikoo et al., 1997) (Figure 6A). Support for this idea has come from the analysis of the fate of oligodendrocyte progenitors that either lack or express elevated levels of p27. Forced overexpression of p27 in progenitor cells inhibits CDK activity and elicits premature cell cycle arrest, even in the presence of mitogen (Tikoo et al., 1998) (Figure 6B). However, these growth-arrested cells do not express oligodendrocyte differentiation markers, apparently separating the requirements for cell cycle arrest from those for cell differentiation. Conversely, oligodendrocyte progenitor cells obtained from mice lacking p27 and grown in the absence of mitogen proliferate for up to two additional cell divisions (Casaccia-Bonnefil et al., 1997; Durand et al., 1998) (Figure 6C). Moreover, the timing of cell cycle exit appears to be a sensitive response to the level of p27, since progenitors obtained from p27 heterozygote mice exhibit an intermediate proliferative behavior, undertaking at most one additional division (Durand et al., 1998). But the ability of oligodendrocyte progenitors to leave the cell cycle in the absence of p27, albeit in a delayed manner, indicates a contribution of other CDK inhibitors or of separate controls on CDK/cyclin activity. The intrinsic machinery that controls the temporal increase in p27 levels in oligodendrocyte progenitors remains unclear, although in nonneural cells, p27 protein levels and activity appear to be regulated primarily by posttranslational mechanisms (Massague and Polyak, 1995). In C. elegans, studies of heterochronic mutants have provided one example of the way in which intrinsic transcriptional programs that control the timing of cell fate Figure 6. A Contribution of CDK Inhibitors to the Timing of Exit of Oligodendrocyte Progenitors from the Cell Cycle (A) Mitogens control oligodendrocyte progenitor division. Progenitors grown in the absence of mitogen rapidly exit the cell cycle and express differentiated oligodendrocyte markers. Oligodendrocyte progenitors grown with PDGF undergo further rounds of cell division but eventually leave the cell cycle and express oligodendrocyte differentiation markers. The intracellular levels of p27 (green) increase progressively with time in vitro. Exposure of cells to thyroid hormone (or retinoic acid) is necessary for cell cycle exit and oligodendrocyte differentiation in the presence of PDGF, but for simplicity this step is not indicated in the diagram. (B) Early forced expression of p27 in oligodendrocyte progenitors induces precocious cell cycle exit, but cells do not express differentiated oligodendrocyte markers. (C) Oligodendrocyte progenitors isolated from mice lacking p27 undergo one or two additional rounds of cell division but eventually leave the cell cycle and express oligodendrocyte differentiation markers. Adapted from data of Raff and colleagues (1998) and Chao and colleagues (see Tikoo et al., 1997, 1998). O, oligodendrocyte. decisions may also direct cell cycle exit. A set of heterochronic genes, notably lin-4, lin-14, and lin-28, control the normal temporal sequence of cell fate decisions (Slack and Ruvkun, 1997). The lin-14 gene has a key role in this process and encodes a nuclear protein that acts as a transcriptional regulator. High levels of LIN14 are expressed at early developmental stages and are involved in the specification of early cell fates. The assignment of later fates requires a decline in the level of LIN-14 expression (Slack and Ruvkun, 1997). A link between heterochronic gene activity and the basic cell cycle regulatory machinery has been provided by the recent finding that in vulval precursor cells the expression of CKI-1, a Cip/Kip class CDK inhibitor, is activated at a transcriptional level by LIN-14 (Hong et al., 1998). Review 219 The Contribution of Cell Cycle Exit to Neural Cell Differentiation The exit of neural progenitors from the cell cycle is accompanied by the onset of expression of many terminal differentiation markers, but the precise contribution of cell cycle exit to neural cell differentiation remains obscure. In considering how cycle arrest might contribute to the specification of neural fate, we return to the examples of neural cell types discussed above that exhibit key fate restrictions at the time of cell cycle exit. One possibility is that cell cycle exit defines the timing of action of transcription factors that specify neuronal subtype identity. Some evidence that this may be the case has come from studies of motor neuron differentiation. The MNR2 homeodomain protein is able to direct several independent features of somatic motor neuron differentiation when misexpressed in neural tube cells (Tanabe et al., 1998). However, the inductive activity of MNR2 appears to operate within the context of an independent developmental program that controls the time at which neural progenitor cells exit the cell cycle. Thus, downstream genetic targets of MNR2 that are normally restricted in their expression to postmitotic motor neurons can be induced ectopically by MNR2 only after neural progenitor cells have left the cell cycle (Figure 7A). A second possibility is that cell cycle exit markedly restricts the ability of neural cells to respond further to certain extrinsic signals. If this is the case, the fate of progenitor cells may be influenced in an important way by the profile of intrinsic determinants that they express at the time of cell cycle exit, a profile presumably established by the ambient signals to which they are exposed during their final progenitor division cycle. The impact of cell cycle exit on the determination of cell fate may therefore depend on the way in which the profile of extrinsic signaling molecules changes during the final progenitor cell division. If the nature or concentration of extrinsic signals changes markedly over time, an alteration in the time of cell cycle exit could result in a switch in cell fate. If, however, the profile of extrinsic signaling remains constant, then precocious or delayed cell cycle exit would be likely to have a much less pronounced influence on cell fate, only altering final cell number. In many regions of the vertebrate CNS, the time at which specific neurons are generated does appear to be closely related to their final identity. Thus, in the cerebral cortex, those neural progenitors that leave the cell cycle at early times acquire neuronal identities distinct from those of progenitors that become postmitotic at later times (McConnell, 1995; Frantz and McConnell, 1996). A similar link between neuronal birthdate and identity is evident in the generation of spinal motor neurons within the medial and lateral divisions of the lateral motor column (Hollyday, 1983). Motor neurons destined to populate the medial division leave the cell cycle well before neurons that form the lateral division. The distinct identity of these later-born lateral division motor neurons appears to be acquired as a consequence of their exposure to a retinoid signal produced selectively by earlyborn lateral motor column neurons (Sockanathan and Jessell, 1998). In this instance, then, the time of neurogenesis has a profound influence on motor neuron subtype identity because of the marked temporal change Figure 7. Transcriptional Control of Neuronal Properties (A) Transcriptional regulation of neuronal subtype identity is constrained by independent controls on neurogenesis and cell cycle exit. Expression of the homeodomain protein MNR2 in response to Shh signaling is sufficient to induce somatic motor neuron transcription factors. The onset of expression of these proteins appears, however, to be constrained by the proliferative state of neural cells. Lim3, an MNR2 target normally expressed in somatic motor neuron progenitors, can be induced ectopically by MNR2 in progenitor cells. In contrast, Isl1 and HB9, transcription factors normally expressed only in postmitotic motor neurons, can be induced ectopically by MNR2 only in postmitotic neurons. Thus, cell cycle exit appears to be required for the expression of postmitotic motor neuron markers. Adapted from Tanabe et al. (1998). (B) Transcriptional control of distinct neuronal properties in autonomic neurons. Expression of Mash1 in response to BMP2 signaling is sufficient to induce the homeodomain protein Phox2a. Mash1 is also required for the acquisition of generic neuronal properties. Phox2a expression is sufficient to induce c-Ret expression and possibly also controls aspects of neurotransmitter phenotype. Phox2a is insufficient to induce generic neuronal properties. Adapted from Lo et al. (1997). in the profile of extrinsic signals in the environment of the developing neuron. A somewhat different view of the relationship between cell fate and cell birthdate has, however, come from an analysis of neuronal fate determination in Xenopus embryos (Harris and Hartenstein, 1991). Inhibition of neural progenitor cell division in vivo by addition of DNA synthesis inhibitors was reported to have little effect on neuronal fates in the caudal neural tube (Harris and Hartenstein, 1991). More generally, it should now be possible to assess the contribution of the timing of cell cycle exit to the specification of particular neuronal fates through the availability of improved cell type–specific markers and the ability to manipulate directly the core cell cycle machinery. Transcriptional Programs and the Coordination of Neural Phenotype Although analysis of the actions of transcription factors has begun to clarify some of the ways in which intrinsic signals control neural cell differentiation, there are many unresolved issues. First, it remains unclear whether there are common transcriptional programs that control Cell 220 expression of the generic neuronal properties shared by diverse classes of neurons. Second, it is unclear whether the subtype identity of individual neuronal cell types requires the convergent activities of many different genes or can be achieved through the actions of a single dedicated subtype-specific factor. Third, there is uncertainty about the mechanisms used to coordinate the assigment of generic and subtype-specific neuronal properties to individual classes of neurons. In Drosophila, bHLH proteins clearly have a central role in specifying neural precursors, but do they also contribute to the assignment of specific neuronal subtype identities? Members of two different subclasses of bHLH proteins, atonal and scute, appear to induce distinct subtypes of peripheral neurons (Chien et al., 1996; Jarman and Ahmed, 1998). The analysis of neurogenesis in the vertebrate retina has also suggested that expression of the bHLH protein Xath5 biases progenitor cells to a ganglion neuron fate (Kanekar et al., 1997). The striking spatial restrictions in expression of individual bHLH proteins in vertebrates may therefore reflect additional roles in conferring elements of neuronal subtype identity. In addition to the activation of positive regulators such as bHLH proteins, the expression of generic neuronal properties may also require the extinction of negative regulators. A zinc finger protein called neuron-restrictive silencer factor (NRSF) (also known as REST) (Chong et al., 1995; Schoenherr and Anderson 1995a, 1995b) has been suggested to play such a role. NRSF/REST appears to act in nonneural cells as a silencer of neuronal gene expression through its interactions with a conserved regulatory element found in the promotors of many neuronally restricted genes (Schoenherr et al., 1996). NRSF/REST is expressed in most nonneuronal cell types and also in undifferentiated neural progenitors but is excluded from neuronal cells, suggesting that it functions normally to prevent the precocious or ectopic expression of neuronal genes. The extinction of expression of NRSF/REST at the time of neuronal differentiation could therefore permit the activation of regulatory elements that promote stable neuronal gene expression. Support for this model has come from studies in which NRSF/REST function has been eliminated in neural cells (Chen et al., 1998). In the absence of NRSF/REST activity, certain neuronal target genes are derepressed in both neural progenitors and in nonneural tissues. These findings raise the important question of the identity of factors that regulate the time of extinction of expression of NRSF/REST in neural cells. Studies of the actions of transcription factors have recently begun to provide evidence for the existence of proteins that have dedicated roles in directing the differentiation of individual neuronal subtypes. The activity of the homeodomain protein MNR2, discussed earlier, appears sufficient to induce a coherent somatic motor neuron phenotype in neural tube cells (Tanabe et al., 1998). It is likely, therefore, that certain transcription factors expressed during the final division cycle of progenitor cells are sufficient to specify the identity of other neuronal subtypes. How are the transcriptional pathways implicated in the assignment of generic and subtype-specific neuronal properties integrated? The most detailed information on this issue has emerged from studies of the differentiation of neural crest cells into autonomic neurons (Figure 7B). This developmental program appears to require the expression of both Mash1 and the paired type homeodomain protein Phox2a (also known as Arix) (Groves et al., 1995; Morin et al., 1997; Hirsch et al., 1998; Lo et al., 1998). These two transcription factors make distinct contributions to the final repertoire of autonomic neuronal properties. Mash1 appears to be required for the expression of generic neuronal markers and, in addition, induces the expression of Phox2a. In turn, Phox2a induces the expression of the c-ret gene (Figure 7B). In addition, the regulatory regions of genes encoding enzymes involved in catecholamine biosynthesis contain Phox2/Arix–binding sites (Valarche et al., 1993; Zellmer et al., 1995; Swanson et al., 1997; Yang et al., 1998), raising the possibility that aspects of neurotransmitter phenotype may also be controlled directly by Phox2a. Mice lacking Phox2a function exhibit marked defects in the development of central noradrenergic neurons, including the lack of expression of the neurotransmitter synthetic enzyme dopamine b-hydroxylase (DbH) (Morin et al., 1997). From these studies, however, it cannot be excluded that the loss of DbH expression is simply the consequence of the absence of noradrenergic neurons. Nevertheless, it seems likely that Phox2a functions as a direct activator of catecholamine neurotransmitter synthetic enzymes in certain neural cells. The transmitter phenotype of central dopaminergic neurons is not affected in Phox2a mutants but has been suggested to depend on the nuclear hormone receptor Nurr1 (Zetterstrom et al., 1997; Castillo et al., 1998; Saucedo-Cardenas et al., 1998). Similarly, the neurotransmitter phenotype of many CNS neurons in Drosophila appears to be controlled in a selective manner by homeodomain proteins (Johnson and Hirsh, 1990; Thor and Thomas, 1997; Benveniste et al., 1998). Taken together, these results begin to suggest that certain subtype-specific features of neurons, notably neurotransmitter phenotype and neurotrophic factor sensitivity, are controlled by transcriptional pathways distinct from those that regulate more generic aspects of neuronal phenotype. Studies over the past decade have shown that the activation of these and other intrinsic transcriptional programs is initially dependent on extrinsic signaling. But over time, intrinsic programs gradually assume a more prominent role in directing the differentiation of neural cells and, in addition, appear to control temporal changes in the ability of neural cells to respond to extrinsic signals. The temporal interplay between extrinsic and instrinsic programs of cell differentiation lies at the heart of cell fate determination. Extracting further details of such interactions and their regulation by cell cycle exit will be an essential step toward the goal of directing the identity of specific neural cell types in the vertebrate nervous system. Acknowledgments We thank David Anderson, Silvia Arber, Bennett Novitch, Leif Carlson, Jane Dodd, Johan Ericson, Helena Edlund, Chris Kintner, and an insightful referee for helpful discussions and/or critical comments Review 221 on the manuscript. We are also grateful to Kathy MacArthur and Ira Schieren for expert help in preparation of the text and figures. 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