Neurogenesis in the vertebrate neural tube

Int. J. Devl Neuroscience 19 (2001) 161– 173 www.elsevier.nl/locate/ijdevneu

Neurogenesis in the vertebrate neural tube Margaret Hollyday * Department of Biology, Bryn Mawr College, Bryn Mawr, PA 19010, USA

1. Introduction Neurogenesis is the developmental process that generates neurons. Since all the cells, including neurons, are ultimately derived from a single cell, the fertilized egg, a complete account of neurogenesis involves understanding the processes that underlie both the proliferative events that produce neuron progenitors, as well as the events that regulate the withdrawal of cells from the proliferative cell cycle. In the developing vertebrate neural tube, both cell proliferation and withdrawal from the cell cycle are patterned in time and space. Thus, developmental processes that regulate neurogenesis necessarily intersect at one or more levels with those processes that pattern the neural tube. This conclusion was also apparent to Viktor Hamburger more than one half century ago when he wrote ‘Any discussion of the problems of regional and topographic determination, …, must include the determination of mitotic patterns, which preceded and are basic to the patterns of differentiation’ (Hamburger and Levi-Montalcini, 1950). Much progress has been made in recent years about the basic molecular machinery that controls the cell cycle, including cells that proliferate in the vertebrate neural epithelium. Nevertheless, some basic questions remain; How is proliferation regulated in neuronal precursors? When and how do progenitor cells ‘decide’ to withdraw from the proliferative cycle? Do environmental cues regulate proliferative behavior, or is there evidence that withdrawal from the cell cycle is controlled by an underlying clock-like mechanism? Does the decision to withdraw from the cycle require changes in the orientation of the mitotic spindle? Since neurons are a diverse population of post-mitotic cells, varying with respect to a number of phenotypic characteristics including the location, size and shape, neurotransmitter and projection patterns; we also want to understand how those characteristics are * Tel.: +610-526-5099. E-mail address: [email protected] (M. Hollyday).

related to the processes of neurogenesis per se. For example, when during neurogenesis is a phenotypic character specified? To what extent is cell fate specified prior to withdrawal from the proliferative cell cycle? Are the events that regulate neuronal differentiation initiated only after a cell withdraws from the cycle? What cues and molecular pathways might regulate these processes? This essay will address the questions focusing on the cells generated in the ventricular epithelium of the vertebrate neural tube, contrasting our knowledge about these events in the spinal cord of birds and mammals, with what is known about other regions of the neural tube. 2. Structure of the neural epithelium When the neural tube first forms, its walls are composed of bipolar shaped cells, each of which spans the entire width of the tube during interphase. The cells in the walls of the neural tube are taller than the cells that form either the floor plate or the roof plate. Only cells in the walls of the neural tube have descendents that give rise to neurons. Floor plate and roof plate cells secrete proteins that provide important patterning cues for neural tube development (see below), but they do not participate directly in neurogenesis. Neuroepithelial cells are morphologically polarized (Chenn et al., 1998). The basal region terminates in a flattened endfoot that contacts basal lamina at the periphery of the tube. The apical end of each neuroepithelial cell abuts the central lumen or ventricle of the neural tube; the surface next to the ventricle is typically folded, and it has a single cilium. Cells are anchored to one another in the apical regions by a variety of specialized junctions (Aaku-Saraste et al., 1996; Bancroft and Bellairs, 1975; Schoenwolf and Kelley, 1980). Junctional contacts along lateral cell surfaces are much less extensive (Nagele and Lee, 1979). In contrast to the apical ends of neuroepithelial cells, the basal endfeet are not connected by specialized junctions (Hinds and Ruffett, 1971; Rodriguez-Boulan and Nelson, 1989).

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3. Interkinetic nuclear migration The nucleus of a neuroepithelial cell moves within the cylinder of cytoplasm, back and forth across the wall of the tube in a process called interkinetic nuclear migration (Chenn and McConnell, 1995; Frishell et al., 1993; Sauer, 1935, 1936). This oscillatory movement depends upon the presence of cytoplasmic microtubules that occur singly or in bundles oriented parallel to the cell’s long axis during interphase. Agents that disrupt microtubules reversibly block interkinetic nuclear migration (Messier, 1978). The region of the neural epithelium that defines the to-and-fro movements of nuclear migration is called the ventricular zone (Boulder Committee et al., 1970). A key feature of interkinetic nuclear migration is that nuclear position varies in relationship to the phases of the cell cycle. Mitotic figures are found only adjacent to the lumen of the neural tube. Cells in S-phase have nuclei located in the outer half of the neural epithelium. This was first deduced by Sauer (Sauer, 1935, 1936) from measurements of the nuclear volume, and has been demonstrated directly by many investigators using either [3H]-thymidine (Fujita, 1962, 1963; Sauer and Walker, 1959; Sidman et al., 1959) or its analogue 5-bromo-2-deoxyuridine (BrdU) (Miller and Nowakowski, 1988). Both of these tracers are taken up by cells and incorporated into newly synthesized DNA, and can be detected in tissue sections. As cells leave the S-phase and enter G2, their nuclei move rapidly towards the ventricular surface, they withdraw their basal process, and begin to round up (Hayes and Nowakowski, 2000; Langman et al., 1966; Nagele and Lee, 1979). Their microtubules become fewer and shorter, losing their apical to basal organization. The luminal surfaces become broader and smoother than they were during G1 and S-phase. The apical adherens junctions and the microfilaments forming the circumferential apical bundle persist throughout G2 and during all phases of the mitosis. Cells become nearly spherical by late prophase and remain at the lumen until late telophase. Many investigators have noted that the majority of mitotic spindles are oriented parallel to the surface of the central canal, especially when the neural epithelium is expanding. After daughter cells separate during telophase, they commonly remain connected to each other by a thread of cytoplasm. Nagele and Lee (1979) thought that the persistence of the threads after completion of mitosis and their variation in length implied that daughter cells were displaced with respect to each other in the neural epithelium following mitosis. This inference has since been demonstrated by clonal analyses of labeled neuroepithelial cells (Fraser et al., 1990; Leber and Sanes, 1995; Mathis et al., 1999).

After a cell completes mitosis and cytokinesis, the basal process is re-extended towards the basal lamina, and its nucleus migrates basally. There are no specific markers for cells in G1, but nuclear size differences and the absence of S-phase markers permit identification of these cells in tissue sections. The nuclei of neuroepithelial cells in G1 are distributed throughout the width of the ventricular zone, but are most abundant in the inner half. Interkinetic nuclear migration towards the periphery during G1 is slower than the reverse migration during G2 (Hayes and Nowakowski, 2000). DNA synthesis takes place when nuclei are positioned in the peripheral half of the neural epithelium. At the end of S-phase, during G2, nuclei migrate back to the ventricular surface. A cell preparing for mitosis withdraws its basal endfoot and rounds up prior to cell division. After division, each of the daughter cells either repeat or exit the cell cycle. Cells that remain in the cell cycle elongate and re-establish contact with the basal surface, thus rejoining the pseudostratified neural epithelium. Those daughters that exit the cell cycle withdraw apical endfoot attachments and move basally through the neural epithelium. Post mitotic cells accumulate peripherally to the replicating neuroepithelial cells. Cells that have exited the mitotic cell cycle are said to be in G0 and are considered ‘born’. The region of cell accumulation is called the intermediate zone and is where postmitotic neurons commence differentiation; the area of the neural tube that is free of neuronal nuclei is called the marginal zone (Boulder Committee et al., 1970).

4. Neurogenesis is patterned in space and time

4.1. Neuronal birthdates mark when neurons exit the cell cycle Neurons are cells that have withdrawn from the mitotic cell cycle; they are said to be permanently in G0. Consequently, one way vertebrate neurons can be characterized is by their ‘birthdate’. In practice, a neuron’s birthdate is not defined as the time when it withdraws from the cell cycle, rather the birthdate is defined as when a neuron completes its final round of DNA synthesis before its terminal mitosis. Markers of Sphase, such as [3H]-thymidine or BrdU, are used to label cells undergoing DNA replication. In mammals, these markers are typically available to the embryo for only a limited period of time estimated from 2 to 7 h (Hayes and Nowakowski, 2000), because the maternal tissues metabolize the tracer and clear it from the maternal/fetal circulation. Only neuron progenitors in S-phase during the time the tracer was available to the embryo incorporate the tracer. If those progenitors withdraw from the cell cycle after the next mitosis,

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there is a high probability that they are heavily labeled; neurons that re-enter the cell cycle and undergo additional rounds of DNA synthesis will be less heavily labeled. In contrast, avian embryos develop in a closed system, the egg, independent of a maternal circulation. Hence, precursors of DNA remain available for incorporation for a prolonged period of time. Birthdating neurons in a cumulative labeling protocol involves determining when a neuron or population of neurons first fails to incorporate labeled DNA precursors. This time defines when neurons have completed their terminal round of DNA synthesis. Although the methods for determining neuron birthdate differ between birds and mammals, the timing of the final round of DNA synthesis can be ascertained with precision in both cases. There is good evidence that cell replication and withdrawal from the cell cycle occur at defined times during development. In addition, the timing of these events varies between regions of the neural tube (reviewed in Jacobson, 1991). Typically in vertebrates, neurons in the hindbrain are the first to withdraw from the mitotic cycle, followed in close succession by neurons of the spinal cord and ventral mesencephalon. With some notable exceptions (e.g. Rohon Beard cells), the first-born neurons contribute to ventral brain structures (e.g. motor nuclei) whereas dorsal structures that perform sensory and integrative functions are populated by neurons produced later in development. Neuron proliferation in forebrain structures begins later than in the other regions and also continues for longer periods than the neurons in the brainstem and spinal cord. Neurogenesis in the mammalian neocortex and cerebellum continues longer than in other places; these may be regions where changes in the regulation of the cell cycle have contributed to the generation of neuronal diversity and evolutionary changes (Caviness et al., 1995; Kornack and Rakic, 1998).

4.2. Viktor Hamburger described regional differences in cell proliferation Before the availability of S-phase markers, studies of neurogenesis were based on histological observations of mitotic figures. One of those papers was published by Viktor Hamburger in 1948. Its title was ‘The mitotic patterns in the spinal cord of the chick embryo and their relation to histogenetic processes’. In that paper, he provided good evidence that cell proliferation is regulated in the spinal cord on the ventral to dorsal axis. Hamburger quantified differences in the density of mitotic figures (defined as the numbers of mitoses/unit area) between the basal and alar plate regions of the spinal cord. He reported that they changed with the developmental age. In the youngest

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embryos, the mitotic density of the basal plate was higher than the mitotic density of the alar plate. This pattern was reversed at the older developmental stages. The increased mitotic density in the basal plate at the earliest developmental stage was correlated with the earlier appearance of the motor neurons in the ventral region of the spinal cord. Similarly, the later development of the interneurons and sensory relay neurons in the dorsal region of the spinal cord was correlated with the increased mitoses in the alar plate observed at the older developmental stages (Hamburger, 1948). Hamburger’s findings provided an explanation for the previously observed ventral to dorsal gradient of spinal cord development and maturation; it could be attributed to quantitative differences in the patterns of cell proliferation in the neural epithelium of the neural tube at different developmental times. He concluded that ‘‘… fundamental structural differences exist in the basal and alar plates and they have their roots in the fundamental differences of the mitotic patterns which were analyzed above’’ (p. 277). In addition, Hamburger also speculated that ‘‘(t)he differentials in mitotic activity in the different regions of the spinal cord, … would be caused by local and temporal differences in the agents which activate mitosis’’ (p. 278). Although Hamburger’s basic observations were confirmed and extended (Corliss and Robertson, 1963), their interpretation remains unclear to this day. One modern interpretation of Hamburger’s results is that differences in mitotic density reflect differences in the duration of the cell cycle in those regions (Lee et al., unpublished observations). Assuming that the duration of M-phase remains relatively constant, differences in mitotic density might reflect differences in the proportion of the total cell cycle devoted to M-phase. A higher mitotic density in one region compared with another implies that the overall duration of the cell cycle in the region of higher mitotic density is shorter than the region with a lower mitotic density. A second interpretation of Hamburger’s mitotic density data is that the basal and alar plates had different proportions of proliferative cells, or in modern terminology, that the growth fractions of these regions were different. Yet another possible explanation of those findings is that regions of elevated mitotic density indicate cells with proportionately longer M phases (a longer TM). Only a more detailed analysis of cell cycle parameters can indicate which if any of these possible explanations is correct at particular stages of development. The processes that control and coordinate patterns of neurogenesis are still not clearly understood. The remainder of this review addresses some of the issues involved in understanding this process in the vertebrate neural tube.

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4.3. Cell lineage and neuronal cell fate Cell lineage studies have revealed that progenitors in the proliferative neural epithelium are for the most part multipotent up until close to their final mitosis (Galileo et al., 1990; Golden and Cepko, 1996; Gray et al., 1988; Leber et al., 1990; Turner and Cepko, 1987; Walsh and Cepko, 1992). Committed progenitors appear to be the exception, not the rule in the vertebrate neural tube (Luskin et al., 1988; Price and Thurlow, 1988). Examples of committed progenitors have been found in rodent cerebral cortex. These cells proliferate in a secondary proliferative zone, the subventricular zone, and give rise to astrocytes. Lineage tracing studies of cells in the chick spinal cord have been done using recombinant retroviruses carrying the gene for b-galactosidase (Leber et al., 1990). The stages when progenitor cells were infected with virus preceded and included the times when motor neurons first withdraw from the mitotic cycle. Clones consisting of multiple cell types including motor neurons, interneurons, glia and ependymal cells were observed most commonly. It is particularly significant that Leber and his colleagues found clones containing motor neurons, interneurons and glia even when the virus was injected at stages 17 – 18. This is after some motor neurons have already completed their terminal round of DNA synthesis, and only one or two cell cycles before all the motor neurons have exited the cell cycle. This finding essentially excludes the possibility of committed motoneuron progenitors in the neural epithelium and is consistent with the conclusion that cell fate of this population is determined near or after the time cells withdraw from the cell cycle. Multicellular clones containing only motor neurons were observed only infrequently, and when they were, the clonally related motor neurons were widely dispersed within the ventral horns, indicating that they belong to more than one motor pool. This observation is also consistent with the conclusion that important aspects of phenotype determination occur after a cell withdraws from the mitotic cell cycle.

4.4. Patterning the spinal cord axes Inductive signals secreted by embryonic organizing centers are responsible for organizing the dorso-ventral axis of the spinal cord (reviewed by Eisen, 1999; Graham, 1997; Lumsden and Krumlauf, 1996; Tanabe and Jessell, 1996). Ventralizing signals from the notochord and floor plate (Ericson et al., 1992; Roelink et al., 1994, 1995; van Straaten et al., 1985; Yamada et al., 1991, 1993), together with dorsalizing signals from the surface ectoderm and roof plate (Dickinson et al., 1995; Liem et al., 1995) are the primary signals that specify the dorso-ventral axis of the spinal cord. A secreted

protein, sonic hedgehog (SHH) is both sufficient and necessary for the ventralizing activity of the notochord and floorplate (Chiang et al., 1996; Echelard et al., 1993; Marti et al., 1995; Roelink et al., 1994, 1995). The dorsalizing signals are members of the TGF-b family; BMP-4, BMP-7, dorsalin and activin are candidates for the dorsalizing signal (Liem et al., 1995, 1997). Together these inductive signals regulate the expression patterns of transcription factors Pax3, Pax6, Pax7, Dbx2, Irx3, Nkx2.2 and Nkx6.1 in partially overlapping domains in the spinal cord proliferative ventricular epithelium (Briscoe et al., 1999, 2000; Ericson et al., 1997; Goulding et al., 1993). When do the dorso-ventral axes of the spinal cord become fixed? Experiments to address this question involve rotations of the neural tube in relation to the surrounding tissue. Rotations of the chick spinal cord on its dorso-ventral axis at stages 12–14 (Hamburger and Hamilton, 1951), result in a partial axis reversal or respecification, as assayed histologically (Steding, 1962). However, if the notochord was included in the rotation, there was little sign of axis reversal. These results can be explained by recent work showing that SHH is secreted by the notochord. The inductive signals responsible for signaling the dorso-ventral axes are both expressed before neural tube closure and during early neural tube stages when the dorso-ventral axes remain undetermined. Signals derived from the paraxial mesoderm determine features of the anterior posterior axis of the spinal cord (Ensini et al., 1998; Fukushima et al., 1996) including the position of the brachial and lumbar enlargements. The signals must also act to determine the proliferative properties of the spinal cord, and they do so progressively along the rostro-caudal axis of the spinal cord. Regions of the spinal cord giving rise to the brachial and lumbar enlargements generate more motor neurons overall than do cervical or thoracic segments (Oppenheim et al., 1989; Sockanathan and Jessell, 1998). Once specified, the segmental properties of the spinal cord are no longer sensitive to external cues. Grafts of the brachial spinal cord segments into either the cervical or thoracic regions at stages 10–13 altered the expression of Lim family homeodomain proteins in accord with the transplanted location, not the origin of the spinal cord segments. Consistent with this observation are the results of rotation experiments. When lumbar spinal cord segments were rotated about the anterior –posterior axis at stage 13 or 14 (Hamburger and Hamilton, 1951), the result was a respecification of motor pools supplying two thigh muscles as assayed by their projection patterns (Matise and Lance-Jones, 1996). Taken together, these results indicate that cues external to the neural tube can influence its axial differentiation. Furthermore, there is a correlation between the loss of responsiveness to patterning cues external to

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the spinal cord and the time when motor neurons first withdraw from the mitotic cycle (Hollyday and Hamburger, 1977).

4.5. Combinations of transcription factors delineate regions of the proliferating neural epithelium and neuronal subtypes Proliferating cells express a number of transcription factors that act in combinatorial ways to specify other transcription factors after withdrawal from the cell cycle. More than a dozen homeobox genes including members of the Lim homeobox family of genes, islet1, islet2, and Lim3 have been identified as determinants of neuronal phenotype in the spinal cord alone (Arber et al., 1999; Briscoe et al., 1999, 2000; Burrill et al., 1997; Ericson et al., 1992, 1997; Sharma et al., 1998; Tsuchida et al., 1994). Nearly all of the transcription factors that define subsets of motor neurons or interneurons are expressed in post-mitotic cells exclusively. MNR2, a homeobox containing transcription factor is one of a few genes that is expressed both in progenitors and in postmitotic motor neurons (Tanabe et al., 1998). Ectopic expression of MNR2 resulted in ectopic expression of Isl1, Isl2 and HB9, characteristic motoneuron markers. Interestingly, although both the mitotically active and post-mitotic cells expressed MNR2, only post-mitotic neurons expressed the specific motoneuron markers. This suggest that the relationship between cell proliferation and withdrawal from the cell cycle was not perturbed by the MNR2 overexpression, and that MNR2 acts within the context of a program of neurogenesis. What regulates the withdrawal of motoneuron progenitors from the proliferative cell cycle and inititates cell differentiation? What marks a cell for withdrawal from the mitotic cycle? Are there changes in the phases of the cell cycle that provide relevant information?

4.6. Measurements and changes in cell cycle parameters during de6elopment Investigators have estimated the duration of the total cell cycle and its components using a variety of techniques. In general, estimates of total cell cycle duration (TC) have ranged from 5 to 18 h in mice, rats and birds (Alexiades and Cepko, 1996; Fujita, 1962; Kaufman, 1968; Martin and Langman, 1965; Polleux et al., 1997; Takahashi et al., 1993, 1995; Waechter and Jaensch, 1972; Wilson, 1973). Estimates of TC of the same region of the same species support the conclusion that there is a general lengthening of the cell cycle with increasing developmental age. However, there is little agreement about which phase or phases of the cell cycle lengthen to produce the overall increase. For example, Takahashi et al. (1995) have found in the developing mouse

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somatosensory cortex that the increased cell cycle time (TC) can be accounted for primarily by an increase in the length of G1 (TG1). The duration of S-phase (TS) and the duration of G2 plus Mitosis (TG2 + M) both remain constant. Waechter and Jaensch (1972) and Wilson (1973) came to a similar conclusion for the developing rat cortex and chicken optic tectum, respectively. These findings would point to G1 as the phase of the cell cycle where important regulatory changes take place. In contrast, in the rat retina, TS increased with advancing developmental age, although the proportion of the entire cycle devoted to S-phase remained relatively constant (Alexiades and Cepko, 1996). Differences in TC between different regions of the same embryos (Dehay et al., 1993; Polleux et al., 1997; Reznikov and van der Kooy, 1995) and between the ventricular zone and the subventricular zone (Haydar et al., 2000; Takahashi et al., 1995) have also been reported. An interesting difference has been reported in the developing cortex of the fetal rhesus monkey. Kornack and Rakic (1998) estimated the cell cycle duration in the proliferative cerebral ventricular zone of fetal rhesus monkeys and found that cell-cycle durations were as much as five times longer in monkeys than the times reported in rodents and birds. In addition, they observed a lengthening of TC from approximately 24 h at the beginning of cortical neurogenesis to more than twice that at a later period. By midpoint during neurogenesis, the estimated cell cycle time had lengthened to approximately 54 h, while at the end of neurogenesis, they found that TC had shortened to approximately 27 h. Kornack and Rakic have suggested that evolutionary modifications of the duration and number of progenitor cell divisions have contributed to both the expansion and the laminar elaboration of the primate neocortex. Taken together, the results suggest that the cell cycle is regulated differently in different regions of the nervous system and at various times during development. The significance of these observations remains a mystery at present but it is tempting to speculate that they reflect the underlying differences in the developmental potential of the various regions, and perhaps their evolutionary potential as well. If the regulatory processes that control progression through the various stages of the cell cycle including withdrawal from the cycle act to increase or decrease the probability of any given event, even slight shifts in probabilities could produce dramatic changes in net outcome.

4.7. Cell cycle: underlying mechanisms Progression through the cell cycle depends on the sequential formation, activation, and subsequent inactivation of a series of cyclins and cyclin-dependent kinase (CDK) complexes (reviewed Sherr and Roberts, 1999;

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Zhang, 1999). The transition from G1 to S involves the activation of several E2F transcription factors that induce the expression of genes necessary for the initiation of S-phase. During the first part of G1, E2F transcription factors are kept in a transcriptionally inactive state because they are associated with the retinoblastoma tumor suppressor gene product pRb. Phosphorylation of pRb protein by cyclin-dependent kinases releases E2F from inhibition and transcription of E2F-dependent genes triggers progression into and through S-phase. Cyclin-dependent kinases cdk2, cdk4 and cdk5 are expressed during G1. They interact with cyclins D1, D2, D3 and E to hyperphosphorylate pRb and release free E2F. The transition from G2 to M is controlled by the cyclin-dependent kinase cdc2, and its regulatory subunits, cyclins A, B1 and B2. Control of G1/S transition is mediated by D-type cyclins thought to be induced by mitogenic signals. D-type cyclins complex with Cdk4 or Cdk6 to carry out the initial phosphorylation of pRb, leading to the de-repression of cyclin E. The activity of cyclinE-Cdk2 drives the cell into S phase. In mammals, there are three D-type cyclins (D1, D2 and D3) and they assemble combinatorially with at least two different CDK subunits, CDK4 and CDK6. The activity of these cdks is positively regulated by their mitogen-dependent assembly with D-type cyclins and by subsequent phosphorylation of the assembled holoenzyme by the CDK-activating kinase CAK (Morgan, 1995). The major function of the D-type cyclins is to provide a link between mitogenic cues and the potentially autonomous cell cycle machinery. Superimposed upon this core circuit are the Cdk inhibitors (CKIs) that are thought to be induced by negative or antimitotic growth signals. Induction of CKIs will prevent phosphorylation of pRb by Cdks, and keep the cells in G1. Cells kept permanently in G1 are said to be in G0. Hence, proteins that interact with the cell cycle machinery to prevent S-phase entry are good candidates for involvement with neuronal differentiation. The CKIs have been assigned to one of two families based on their structure and CDK targets: Inhibitors of CDK4 (Ink4 proteins whose members are p17Ink4a; p15Ink4b, p18Ink4c, p19Ink4d), and the Cip/Kip family (whose members are p21Cip1, p27Kip1, and p57Kip2). The INK4 family specifically inhibits Cdk4 and Cdk6, whereas the KIP/CIP family inhibits all the Cdks involved in the G1/S transition. The signals that induce either the cyclins or CKIs remain to be identified.

4.8. The molecular machinery for cell cycle progression is expressed in the de6eloping neural tube Expression of genes whose proteins control progression of the cell cycle has been demonstrated in various

regions of the developing vertebrate nervous system. B cyclins and their associated cyclin dependent kinase cdc-2 (also called CDK1) regulate the transition from G2 into mitosis. As one might expect, these genes are expressed in the ventricular zone of both the spinal cord and neocortex (Delalle et al., 1999; Tsai et al., 1993; Zhao et al., 1995). Expression of cdc-2 is downregulated in the developing brain when the cells withdraw from the ventricular zone and differentiate in the intermediate zone (Hayes et al., 1991). Cyclin E which completes the G1 to S transition and is active during the S-phase, and its associated kinase CDK2 are also expressed in proliferating neuroepithelial cells (Delalle et al., 1999; Lee et al., 1996; Tsai et al., 1993; Zindy et al., 1997). CDK2 protein levels decline with the developmental age. The D cyclins that regulate the first part of G1 exhibit a more complex pattern of expression. Cyclin D1 is expressed in proliferating cells (Miyazawa et al., 2000; Zhao et al., 1995), whereas Cyclin D2 is expressed in the marginal zone of the spinal cord (Zhao et al., 1995) and in cerebellar granule cells as they make the transition from proliferating to non-proliferating cells (Ross et al., 1996; Ross and Risken, 1994). Thus, D cyclins may have played a role in both proliferation and cell differentiation. The CDK partners of the D cyclins, CDK4 and CDK6, are expressed in proliferating neural epithelium (Lee et al., 1996; Zindy et al., 1997). These proteins were detected in the mouse from E14.5 until postnatal day 7, but were absent in the adult brains. Disruption of a D2-cyclin, which is normally expressed in post-mitotic cerebellar granule cells, resulted in a reduced cerebellum and loss of granule cells and stellate interneurons (Huard et al., 1999). A pulse of BrdU was used to identify cells in the S-phase. The investigators also found a reduced labeling index in embryos lacking a functional D2-cyclin. One interpretation of this result is that the absence of D2 slows the cell cycle. Another consequence of cyclin D2 disruption was increased apoptosis. The targets of the D cyclins and their associated CDKs, the retinoblastoma protein (pRb), and two related proteins, p130 and p107 are also expressed in the proliferating neuroepithelial cells of the spinal cord and brain (Jiang et al., 1997; Zhao et al., 1995). Retinoblastoma protein and p130, but not p107 are also expressed in post-mitotic cells in the brain and spinal cord. Hypophosphorylated pRb binds to the E2F family of transcription factors preventing transcription of genes necessary for S-phase. As expected, E2F family members are also expressed in proliferating neural epithelial cells (Dagnino et al., 1997; Loiseau et al., 1997; Zhao et al., 1995). Different E2F family members are expressed in different regions and a few are also expressed in post-mitotic cells (Dagnino et al., 1997). The diversity and functional redundancy of these important compo-

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nents of the cell cycle machinery may be important for differences in the competence of cells in different regions to respond to various patterning signals that regulate neuronal determination and differentiation (Classon et al., 2000).

4.9. Regulation of the neuroepithelial cell cycle Perhaps the more interesting question concerns expression of cell cycle regulatory genes that might inhibit progression of the cell cycle, and thus function as neural epithelial cells undergo the transition from progenitor to post-mitotic cell. Do cells withdraw from the mitotic cell cycle because the mitogenic stimuli fall below some level necessary for inducing the D cyclins? Or is the G1 to S transition actively repressed by the expression of inhibitor? Is there some other mechanism that regulates exit from the cell cycle such as one that controls the orientation of the mitotic spindle? Or one that regulates cell adhesive interactions associated with the adherens junctions at the apical regions of neural epithelial cells? A number of proteins with mitogenic actions on the ventricular neural epithelium have been identified including Wnt1 (Dickinson et al., 1994), fibroblast growth factor (Dono et al., 1998; Ortega et al., 1998; Vaccarina et al., 1999) and Sonic hedgehog. Sonic hedgehog has been shown to have mitogenic effects on CNS precursor cells in vitro (Jensen and Wallace, 1997; Kalyani et al., 1998; Weschler-Reya and Scott, 1999), and in the spinal cord in vivo (Rowitch et al., 1999). Sonic hedgehog protein is normally produced by the notochord and floor plate and distributed in a gradient from its ventral source (Ericson et al., 1997). When expressed ectopically in the dorsal region of the spinal cord, Shh increased the number of BrdU labeled cells in an overgrown ventricular epithelium at 12.5 days post coitum when ventricular epithelial cells are normally proliferating, but not at 18.5 days post coitum when the spinal cord neurogenesis is completed (Rowitch et al., 1999). The proliferative effect of dorsally produced Shh appears to have been limited by another, as yet unidentified regulator of proliferation within the neural epithelium. Another reason cells withdraw from the mitotic cell cycle is because the transition from G1 to S-phase is actively repressed. This model of cell cycle withdrawal has multiple molecular candidates including pRb, and the cyclin dependent kinase inhibitors (CKIs). The activity of pRb depends upon its state of phosphorylation; in its hypophosphorylated state it inhibits the transcriptional activity of E2F family members which are necessary for the G1 to S transition. Retinoblastoma protein can also actively represses promoters of E2F target genes via its ability to recruit histone deacetylase (reviewed Zhang, 1999). Histone deacetyla-

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tion is thought to facilitate the formation of nucleosomes and therefore hinder access to promoters by transcription factors. Thus, hypophosphorylated pRb could function to promote withdrawal from the cell cycle. It is unclear whether pRb functions to initiate withdrawal from the cell cycle, but there is good evidence that functional pRb is important for maintaining cells in a non-proliferative state. The retinoblastoma protein and the closely related gene p130 are normally expressed in both proliferating and in post-mitotic neurons (Jiang et al., 1997; Zhao et al., 1995). In the absence of functional retinoblastoma protein, ectopic mitoses are observed outside the ventricular zone of the spinal cord (Lee et al., 1992, 1994). In normal development, mitotic figures are restricted to the ventricular surface of the neural tube. Genetic disruption of the closely related gene p130 resulted in a different phenotype (LeCouter et al., 1998). Ectopic mitoses were not observed as in the pRb knockout, motor neuron production was disrupted instead. LeCouter et al. (1998) found severely decreased numbers of Isl-1/2 expressing cells in the ventral horn of a somewhat disorganized neural tube. It should be noted that the genetic background of mice was important for this result. In a Balb/cJ genetic background, p130 was essential for normal differentiation of motor neurons, while in C57BL/7 mice disruption of p130 had no visible effects on spinal cord development. Thus, other genes exist that have an epistatic relationship with p130. Exit from the cell cycle during terminal differentiation also requires the inactivation of cyclin dependent kinases by cyclin kinase inhibitors (CKIs). The three members of this family, p21Cip1/Waf1, p27Kip1 and p57Kip2 are all expressed in both proliferating and postmitotic neuroepithelial cells in various regions of the mouse brain (Campagne and Gill, 1998; Delalle et al., 1999; Dyer and Cepko, 2000; Lee et al., 1996; Miyazawa et al., 2000; Zindy et al., 1999), and each is expressed in a dynamic and restricted pattern during development. Progressively increasing levels of p27Kip1 have been correlated with withdrawal from the cell cycle in studies in a number of systems both in vivo and in vitro (Durand et al., 1998; Lee et al., 1996; Miyazawa et al., 2000; Ohnuma et al., 1999). Genetic disruption of the p27Kip1 gene in mice leads to increased numbers of neurons in the brain (Fero et al., 1996; Kiyokawa et al., 1996). Interestingly, there is also evidence that the build-up of p27Kip1 may be regulated by an intrinsic timing mechanism, first proposed by Raff and his colleagues (Durand et al., 1998; Gao et al., 1998, 1997; Raff, 1996). In isolated, cultured ganglion cell precursors, an inverse correlation between BrdU uptake and p27Kip1 expression was observed (Miyazawa et al.,

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2000). Even when cultured in the presence of Sonic hedgehog protein, a potent mitogen for cerebellar granular cells (Weschler-Reya and Scott, 1999), the cells eventually stopped dividing and differentiated, expressing p27 strongly. The INK4 proteins are polypeptide inhibitors of the cyclin D dependent kinases CDK4 and CDK6. Ink4 family members are also expressed in the neural epithelium at times when cells withdraw from the cell cycle and differentiate (Zindy et al., 1997). The p18Ink4c was first expressed in proliferating neuroepithelial cells when cortical neurons switched from a symmetric to an asymmetric pattern of mitosis. This inhibitor was replaced by p19Ink4d in postmitotic cells. In contrast to the situation when p27Kip1 was disrupted, disruption of p19Kip4d did not produce a dramatic phenotype (Zindy et al., 1999). A double knockout of both p19Kip4d and p27Kip1 resulted in ectopic neuronal cell divisions (Zindy et al., 1999), similar to what was observed following disruption of the retinoblastoma gene. This result points to the functional redundancy of some regulatory proteins, and additionally suggests that the mechanism underlying the initial withdrawal from the cell cycle may be different from those maintaining cell cycle arrest.

4.10. Orientation of the mitotic spindle and withdrawal from the cell cycle The orientation of the mitotic spindle with respect to the surface of the ventricle has been proposed to explain the withdrawal of cells from the neural epithelium and thus regulate the production of neurons (Chenn and McConnell, 1995; Langman et al., 1966; Martin, 1967). When the mitotic spindle is oriented parallel to the ventricular surface, the cleavage furrow is perpendicular to the surface. Consequently, each of the daughter cells will receive part of the original network of adherens junctions, and therefore probably remain associated with the neuroepithelial sheet to re-enter the mitotic cell cycle. This type of mitotic division has been called a symmetric division. When the spindles are oriented perpendicular to the epithelial surface instead, the cleavage furrow would be parallel to the surface, and the daughter farthest from the ventricular surface would lose its attachments via the terminal bar network. Such a daughter might be free to migrate out of the neural epithelium and begin differentiation. Although this model has considerable appeal, earlier studies of cleavage orientation in fixed sections through the cerebral ventricular zone have suggested that the vast majority of all cleavages are oriented symmetrically even during peak periods of neuronal production (Smart, 1973; Zamenhof, 1986) (and Hollyday unpublished observations). Whether or not inhibitors of the cell cycle indirectly regulate orientation of the mitotic

spindle, it is clear that they influence the withdrawal of cells from the pseudostratified neural epithelium. It has been proposed that the activation of the Notch signaling pathway by Notch-ligand expressing neighbors prevents cells from undergoing differentiation and that inhibition of this pathway would promote differentiation (Artavanis-Tsakonas et al., 1999; Lo et al., 1991). NUMB protein blocks activation of the Notch signaling pathway and would therefore be expected to promote withdrawal from the cell cycle. If differentiation is blocked by maintaining the neuroepithelial cells in the proliferative cell cycle, one would predict that activation of the Notch pathway would increase the rate of cell proliferation (reduce cell cycle time), and that expression of repressors of the Notch signal would decrease rate of production (increase cell cycle time). It would be interesting to learn what phases of the cell cycle might be regulated by the Notch signaling pathway. Notch and its signaling partners Serrate and Delta are expressed in the proliferating neural epithelium of the chick spinal cord throughout the period when neurons are being born (Myat et al., 1996). Data addressing this model in the vertebrate neural epithelium have been reported (Chenn and McConnell, 1995; Wakamatsu et al., 1999). Chenn and McConnell (1995) reported the asymmetric distribution of Notch1 immunoreactivity in mitotic cells, with Notch 1 selectively inherited by the basal daughter of asymmetrical mitotic division. This is an unexpected result in view of the role that Notch is thought to play in inhibiting neuronal differentiation, not in facilitating the differentiation. However, the findings might indicate that the Notch signaling pathway has additional, as yet undefined roles in mediating the process of differentiation. The presence of NUMB immunoreactivity within the basal cortex of mitotically active neuroepithelial cells has been reported (Wakamatsu et al., 1999). If the cell subsequently undergoes an asymmetrical mitotic cell division, its basally positioned daughter would be in a position to incorporate all of the NUMB protein and initiate differentiation. Presumably, in the case of symmetrical cell division, the concentration of NUMB protein would not interfere with activation of the Notch pathway that is thought to maintain cells in an undifferentiated state.

4.11. When is neuronal cell fate specified? There is good evidence that multiple environmental signals including diffusible signals, activity and experience influence the fate of neural progenitor cells (reviewed Cameron et al., 1998; Edlund and Jessell, 1999; Levitt, 1995). Neuronal precursors express various combinations of signaling molecules and their receptors, and also the combinations of transcription factors, many of which are likely to be involved in specifying

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neuronal cell fate. Interestingly, the vast majority of molecular markers used to define neuronal phenotype are expressed either in proliferating neural epithelial cells, or they are expressed only after cells enter G0 and begin to differentiate. With the exception of the cell adhesion molecules such as NCAM and N-cadherin, it is difficult to find examples of neuronal markers that are stably expressed both before and after withdrawal from the mitotic cycle. Given the dearth of markers for specified, but as yet undifferentiated neurons, how can one decide whether the cell fate decision is made by the progenitor cell, or by the daughter? Perturbation experiments in which progenitor cells are either transplanted into a foreign environment (McConnell and Kaznowski, 1991) or exposed to signals in cell culture (Belliveau and Cepko, 1999; Eagelson et al., 1997) support the conclusion that environmental cues determine neuronal cell fate. Even more importantly, these experiments have shown that there is a critical period for exposure to these cues with respect to phase of the cell cycle. The environmental cues that can specify neuronal cell fate must be experienced within hours of the terminal mitosis, during late S-phase or during G2 to be effective. Exposure to the same cues at other times during the cell cycle does not produce the same change in cell fate. None of these experiments shed light on why progenitor cells withdraw from the cell cycle instead of re-entering S-phase, but if they do withdraw, they subsequently differentiate as if they had received important instructive information about their cell fate just before they were born.

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proportion of cells exiting the cell cycle do so as a consequence of a perpendicular (or asymmetric) mitotic division, and what proportion involves one or more daughters of parallel (or symmetric) mitotic divisions. Obtaining such information would require more extended observations of proliferating neuroepithelial cells in vivo than have been done to date. Another possibility is that the ‘decision’ to withdraw from the cell cycle occurs during G1 and involves one or more of the daughter cells produced from mitotic divisions in which the spindle was oriented parallel to the ventricular surface. In this situation, the properties of the adherens junctions that connect neural epithelial cells at their apical processes would change sometime during G1, presumably in response to whatever signal(s) are responsible for arresting cells in G1. This change would allow the daughter to migrate out of the ventricular zone and enter into the intermediate zone where it continues to differentiate. In Drosophila, adherens junctions are regions in which a number of proteins that regulate cell proliferation and patterning including Notch proteins are localized or concentrated (reviewed in Woods and Bryant, 1993). Although there is little evidence at present to suggest that the cell cycle of the vertebrate neurepithelium can be regulated via molecules associated with adherens junctions, such a model is consistent with what is known. The hypothesis connects the events of cell proliferation to cell fate determination and differentiation (see Fig. 1).

5. Conclusions

4.12. What marks a cell for withdrawal from the mitotic cycle? It is generally accepted that the decision to exit the cell cycle is made during G1, and the best candidates for carrying out this decision appear to be proteins whose function is to inhibit progress through the cell cycle, arresting cells in G1 and preventing their entry into S-phase. This conclusion presents an interesting problem with respect to the proposed relationship between changes in the orientation spindle that accompany neurogenesis in the cerebral cortex (McConnell, 1995), and by extension to neurogenesis elsewhere in the neural tube. The events that determine the orientation of the mitotic spindle must occur after replication of the centrosome during S-phase and prior to mitosis. If withdrawal from the cell cycle involves a change in the orientation of the mitotic spindle from parallel to perpendicular to the ventricular surface, then the ‘decision’ is made prior to the terminal mitosis, probably in late S or in G2. Thus, the ‘decision’ to withdraw from the cell cycle based on a change in spindle orientation cannot be the same event as one that arrests a daughter cell in G1. It would be interesting to know what

The close relationship between withdrawal from the cell cycle and neuronal differentiation has been appreciated for a long time. One molecular candidate linking these processes has been recently identified in the Xenopus retina. Ohnuma et al. (1999) demonstrated that at least one cyclin kinase inhibitor has two different functional activities. p27Xic1 has both the expected kinase inhibiting activity and also an inducing activity. Expression of p27Xic1 in developing Xenopus retinas was observed at the time cells normally withdraw from the mitotic cell cycle and begin differentiation. Surprisingly, over-expression of p27Xic1 not only caused withdrawal from the cell cycle as would be expected it also changed the pattern of differentiated cells produced. Over-expression of p27Xic1 increased the absolute number of Mu¨ller cells produced in the retina while at the same time reducing cell proliferation of all other cell types. To demonstrate that the differentiation of additional Mu¨ller cells could be attributed to p27Xic1 activity and was not simply an indirect consequence of the cell cycle arrest, the cell cycle was arrested with dominant negative forms of both Cdk2 and cdc2. Disruption of the activity of Cdk2 blocks the transition from G1 to S and

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did not induce Mu¨ller cells; neither did the disruption of cdc2, which blocks the G2/M transition. If other cyclin kinase inhibitors also have dual functions, they might act as links between cell cycle exit and regulators of cell fate. One pathway that might link the two processes is the Notch/Delta signaling pathway. Consistent with this suggestion is the observation that manipulated over-expression of p27Xic1 together with a constitutively active form of the intracellular signaling domain of Notch induced even higher levels of Mu¨ller cell induction (Ohnuma et al., 1999). The importance of understanding the relationships between neuronal patterning, regulation of neuronal production via the mitotic cell cycle, and regulation of neuronal differentiation was recognized by Viktor Hamburger more than one half century ago. Although good progress has been made in this direction, much remains to be learned. In closing this article written in honor of Viktor Hamburger, the author would like to express her appreciation for his wisdom and foresight, for his kind and wise mentorship along the journey of exploration and discovery, and for his own efforts to

Fig. 1. Summary diagram illustrating some of the important life history events during neurogenesis. Drawing is of a portion of the wall of an embryonic neural tube showing individual cells at various stages of the cell cycle. The ventricular surface is at the bottom of the diagram, the pial surface is at the top. The nuclei of proliferating cells undergo interkinetic nuclear migration that is coordinated with phase of the cell cycle. Nuclei move away from the ventricular surface toward the pial surface during G1 and undergo DNA synthesis (S) in the outer half of the ventricular zone. They return to the ventricular surface during G2 where they undergo mitosis (M). Neural epithelial cells are connected to one another at their apical ends by adherens junctions (open arrowheads). Proliferating cells acquire information from their environment during late S-phase and during G2 that specifies cell fate. After completing mitosis and re-entering G1, cells ‘decide’ whether to exit the cell cycle and differentiate, or they ‘decide’ to re-enter the cell cycle. A ‘decision’ to exit the cell cycle must be accompanied by the loss of adhesive connections with other cells. Adapted from Jacobson (1991) based on Sauer (1935).

find answers to many important problems that still confront contemporary developmental neurobiologists.

Acknowledgements I thank Marion L. Pepper for stimulating discussions and for help in preparing the manuscript, Steve Leber and Josh Sanes for their input, and Terri Freedman and Betsy Reese in the Bryn Mawr College Library for their help in obtaining the reference materials.

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