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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.
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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.
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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
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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.,
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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).
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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
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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. We
apologize for the inability to cite all relevant work, a consequence
of limitations on the length of the article. T. M. J. is an Investigator
of the Howard Hughes Medical Institute.
Mandel, G. (1995). REST: a mammalian silencer protein that restricts
sodium channel gene expression to neurons. Cell 80, 949–957.
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