Vertebrate neural progenitor cells: subtypes and regulation Sally Temple 1 and Xueming Qian

Several advances have been made recently in characterizing neural progenitor cells. In vertebrates, multipotential stem cells have been demonstrated in the developing forebrain both in vitro and in vivo, and a class of stem cells has been identified in the adult CNS. Factors that regulate the proliferation and differentiation of subtypes of neural progenitor cells have also been described. In invertebrates, progress has been made in identifying genes involved in neural progenitor cell specification, cell-fate choices and regulation.

Address Department of Pharmacology and Neuroscience, Albany Medical College, 47 New Scotland Avenue, Albany, New York 12208, USA 1 e-mail: Sally_Temple@ccgateway.amc.edu

Abbreviations A-P anterior-posterior bFGF basic FGF Dil dioctadecyl-tetramet hyl-indocarbocyanine perchlorate D-V dorsal-ventral E embryonic day EGF epidermal growth factor FGF fibroblast growth factor GABA ~-aminobutyric acid O-2A oligodendrocyte type-2 astrocyte SVZ subventricular zone VZ ventricular zone

we discuss advances made over the past year towards our understanding of neural progenitor cells, focusing on development of the mammalian CNS.

C l a s s e s o f n e u r a l p r o g e n i t o r ce l ls Despite having similar morphologies, germinal cells in the CNS ventricular zone (VZ) consist of multiple types, defined on the basis of two criteria: first, what neural cells they can generate and second, their proliferative potential. These features have been assessed by following the fate of single identified cells in vivo and in vitro (for a recent review, see [2]). Considering the first criterion, the two fundamental neural cell types are neurons and glial cells. On this basis, neural progenitor cells fall into three classes: cells that can generate both neurons and glial cells (neuron/glial progenitor cells), progenitor cells that gt~nerate only neurons (neuroblasts), and those that generate only glial cells (glioblasts). Neuroblasts and glioblasts can be muhipotential, generating more than one type of neuron or glial cell, respectively. Alternatively, they may be unipotentiai, generating only one type of progeny; such cells are sometimes referred to as 'precursors', denoting their pre-ordained fate. Figure 1 summarizes these different classes of neural progenitor cells.

Current Opinion in Neurobiology 1996, 6:1 I - I 7

© Current Biology Ltd ISSN 0959-4388

I n t r o d u c t i o n A remarkable feature of the nervous system is its wealth of cell diversity. Neural progenitor cells are the forerunners of these diverse cell types. They generate the numerous neurons and glial cells appropriate for each neural region. In addition, they orchestrate the appearance of different cell types in a precise temporal order, thus playing a part in generating the complex intercellular relationships that characterize the nervous system [1].

To understand better how neural progenitor cells develop, we need to characterize these cells. It is important to determine the cell types that a progenitor cell gives rise to, and how many progeny it generates. On the basis of these criteria, it is now clear that progenitor cells are a heterogeneous population (reviewed in [2]), raising the 'upstream' issue (i.e. what factors specify different neural progenitor cell types). Defining progenitor cells is of course only the starting point to understanding their biology; we need to identify factors regulating key events in progenitor cell development, such as division, differentiation and death, to understand how these cells build the nervous system. In this review,

The types of progenitor cell present in different neural regions at different times are currently being explored. Results thus far demonstrate that different germinal regions are composed of different types of progenitor cell. For example, embryonic retina and spinal cord contain mainly neuron/gila progenitor cells that can maintain their muhipotential nature, even up to the last division in vivo [3,4]. There appear to be no unipotential neuroblasts in these regions; however, glioblasts that generate only astrocytes or only oligodendrocytes have been described in spinal cord [3,5]. In contrast, in cerebral cortex, neuron/glial progenitor cells are rare: the majority of progenitor cells generate clones consisting of neurons, astrocytes or oligodendrocytes (reviewed in [2,6]). Furthermore, most cortical neuroblasts generate only one of the major classes of cortical neuron, either pyramidal or non-pyramidal cells [7]. Likewise, in the cerebellum, neuroblasts in the external granular layer generate solely granule neurons [8], and in the hindbrain, different classes of neuroblasts are also restricted to one neuronal type [9"'].

Production of unipotential progenitor cells allows selective amplification of that neural cell type. Thus, it appears that this mechanism of generating multiple copies of individual cell types is an important feature of cortical, cerebellar and hindbrain development, both in neuronal and glial lineages. However, in retina and spinal cord, it plays a less prominent role in generating classes of neurons (as defined by current markers).

1 2 Development

Figure 1

© 1996 Current Opinion in Neurobiology

Classes of neural progenitor cells present in the developing nervous system. (a) Neuroblasts and (b) glioblasts can be multipotential, generating more than one cell type, or unipotential. (c) Neuron/glial progenitor cells are multipotential and can generate either unipotential or multipotential neuroblasts or glioblasts, which could divide further or differentiate into mature neurons or gila without division. Only a proportion of the possible outcomes are depicted in (c).

The second criterion concerns the proliferative behavior of neural progenitor cells. In some non-neural systems, such as blood and skin, there are sub-populations of germinal cells that are capable of self-renewal, or self-maintenance. These cells have been defined as stem cells on the basis of this property [10]. Their extensive proliferative capacity makes them the basic ancestor cell for the tissue. This cell type is illustrated in Figure 2. In a historical context, the term 'stem cell' has also been used during embryonic development to denote a multipotential ancestor cell, regardless of its mode of division. In this review, the term stem cell will refer more specifically to a self-renewing cell.

Recently, the types of neural progenitor cells present in the developing CNS has been expanded by the definition of classes of stem cells present in the developing forebrain. A single-cell study conducted in vitro revealed a rare class of multipotential cell in embryonic cerebral cortex that can generate large clones of neurons, astrocytes, and oligodendrocytes, demonstrating for the first time that all three major CNS neural cell types could have a common ancestry [11"]. The multipotential cells were shown to self-renew, a defining feature of stem cells (Figure 2). Cells with similar characteristics have since been described in mass cultures of cortical cells [12"]. In addition, a multipotential epidermal growth factor (EGF)-responsive striatal cell described previously was also shown to self-renew extensively and to generate neurons, astrocytes and oligodendrocytes, providing evidence for a similar multipotential stem cell in basal forebrain [13].

There are pros and cons to examining neural progenitor cell characteristics in vivo and in vitro. Studies in vivo provide fate maps showing what cell types progenitor cells make during normal development. However, this does not necessarily tell us the possible fates of that cell given different growth conditions. In vitro studies allow us to examine this issue. However, data acquired in vitro must be viewed as showing what a progenitor cell is capable of, not necessarily what it does. The O-2A progenitor cell is perhaps a sobering example of how important it is to stress this point. This cell, first identified in optic nerve, differentiates into both oligodendrocytes and a specialized astrocyte (the type 2 astrocyte) in vitro (reviewed in [2]). However, its astrocytic product has remained elusive during normal development in vivo--al though it may be generated during pathological states of the adult CNS [14]. Clearly, a marriage of in vivo and in vitro studies is necessary for a full understanding of neural progenitor cell characterization.

How are different classes of neural progenitor cells related? While both stem cells and more restricted (in terms of developmental potential and proliferative potential) neural progenitor cells co-exist, the relationships among them may be understood by drawing an analogy with the hemopoietic system. In blood production, a popula- tion of multipotential stem cells divides asymmetrically, generating restricted progenitor cells that proliferate and differentiate into mature blood cell types [15] (Figure 2).

Vertebrate neural progenitor cells Temple and Qian 13

Figure 2

© 1 gg6 Current Opinion in Neurobiology

Development of multipotential stem cells. Note that stem cells can divide symmetrically tO generate two stem cells or asymmetrically to generate a stem cell and a restricted progenitor cell or a differentiated product. There are a number of points at which exogenous factors can influence this process: first, during division of stem cells; second, by altering the types of cells that stem cells produce; and finally, during division and differentiation of stern cell progeny. Neurons may be produced before glial cells by sequential generation from stem cells.

By analogy, neural stem cells may generate restricted progenitor cells for neuronal or gliat cell types. This model was suggested to explain neural crest development, spurred by the observation of self-renewing stem cells amongst neural crest progenitor cells [16]. However, at that time there was limited evidence for the existence of classes of restricted precursors that could represent the products of these cells. Hence, the importance of a paper published recently that describes a family of restricted progenitor cells in the enteric lineage [17°°]. Interestingly, these enteric progenitor cells exhibit different levels of commitment, suggesting that the restriction in gene expression from a stem cell to a unipotential precursor cell may occur through a series of gradual steps, as has been suggested for the hemopoietic system [18]. Now it remains to firmly draw the lineage line between multipotential neural crest stem cells and these restricted progenitor cells. Similar models have been presented in relation to CNS development, and again co-existence of multipotentiai stem cells and restricted progenitor

cells provides circumstantial evidence for this model [19,20,21"°,22°]. In addition, subcloning of multipotential neural stem cell clones generates restricted progenitor cells for neurons or glial cells, showing that these classes of progenitor cells are directly related in vitro [11°°,23].

Importantly, evidence for this relationship in vivo has now emerged. In a recent retroviral lineage study of progenitor cells in embryonic day (E)-15 and El7 rat cerebral cortex, a large number of identifiable tags were used to examine widely distributed clonal progeny [21°°]. The clones comprised a variety of clustered groups of cells, often placed at a set periodicity. As reported in previous retroviral lineage studies, the composition within any one cluster was usually uniform (i.e. atl the same cell type), whereas it varied between clonally related clusters (i.e. clusters next to each other could be composed of different cell types). This suggests that a migratory, multipotential precursor cell can generate different restricted progenitor cells in vivo. We would like to know the relationship of these cells to multipotential cortical stem cells identified in vitro.

Although a hemopoiesis-like mechanism of cell generation could apply to some regions of the CNS, such as the cerebral cortex, it is not clear how it applies to others, such as spinal cord and retina. In both these regions, multipotential cells are present, and there is some evidence for restricted spinal glial progenitor cells, but not for restricted neuronal progenitors, as described above. It remains to be seen whether the multipotential cells in these regions self-renew and generate the classes of restricted progenitor cells that are present. Interestingly, in the vertebrate retina, there is some evidence for restriction in the repertoire of cell types that a multipotential progenitor cell can generate as development proceeds (reviewed in [24]). Thus, it is possible that common mechanisms underlie the restriction in developmental potency of multipotential cells in all these neural regions.

Stem cells must divide asymmetrically to generate another stem cell and a different daughter ce l l - -a l though not all asymmetrically dividing cells are stem cells. That asymmetric divisions take place in neural progenitor cells has been deduced from studies of cell proliferation kinetics and from patterns of retroviral clones in monkey cortex [22"]. Asymmetric divisions have since been observed directly using confocal imaging of DiI-labelled VZ cells in ferret cortical slices [25"]. Mechanisms underlying asymmetric divisions in neural progenitor cells are being explored. The products of two genes, Numb (a membrane-associated protein) and Prospero (a nuclear protein), are segregated differently to daughter cells during asymmetric neural progenitor cell divisions in Drosophila [26-28]. Pursuit of vertebrate homologues of these genes may help to resolve the mechanisms of asymmetric division in the nervous system. In the VZ, Notch is distributed differentially among daughter

14 Development

cells during asymmetric divisions [25"]. Although Notch acquisition is passive in this case (i.e. not linked to the cell-division machinery [29]), it may play a role in establishing cell asymmetry.

In summary, although muhipotential stem cells exist in some regions of the nervous system, their contribution to CNS development remains to be clarified. Such cells may be important ancestor cells, analogous to Drosophila neuroblasts that, despite the name, are stem cells that generate restricted neuronal and glial progenitor cells [30]. Alternatively, some, or even the majority, of other classes of more restricted progenitor cells may arise independently.

Specification of neural progenitor cells It is not yet known what factors specify different classes of vertebrate neural progenitor c e l l - - for example, what factors confer muhipotency, unipotency, prolifera- tive potential or regional characteristics. In Drosophila, neuroblasts are generated and specified by the actions of numerous genes, including patterning, proneural, and neurogenic genes [31]. In addition, recent evidence points to the action of segment polarity genes, such as gooseberry distal and POU domains genes pdml and pdm2 in specifying fates of individual neuroblasts [32-34].

Neural progenitor cell identity in vertebrates, as in invertebrates, is initially defined by factors generating anterior-posterior (A-P) and dorsal-ventral (D-V) coordi- nates in the embryo. Recently, it has been demonstrated that Sonic Hedgehog, a mediator of patterning in the D-V axis, can specify the fate of ventral neurons along the neural tube: motor neurons in spinal cord [35], dopamin- ergic neurons in midbrain [36], and cholinergic neurons in basal forebrain [37]. The same factor produces different cell phenotypes, perhaps reflecting A-P differences in the responding cells. In addition to influencing neuronal type, signalling from the notochord may induce the appearance of oligodendrocytes of ventral cord [5]. The types of neural progenitor cell influenced by signals released from the notochord are not yet clear.

Refinement of A-P and D-V patterning generates dif- ferent regions of the nervous system. The number of genes found to be expressed in neural progenitor cells in a region-specific manner is growing, and many of these are related to genes involved in Drosophila neurogenesis (reviewed in [38,39]). In the past year, for example, regional expression patterns of two homologues of the Drosophila proneural gene atonal, Math-1 and illath-2, have been described [40,41]. Null mutations of regionally expressed genes are providing more direct evidence for their role. Over the past year, for example, mutations of BF-1 (a homologue of Drosophila forkhead) and Otx-2 (a homologue of Drosophila orthodenticle) have been reported, and both result in lack of specific CNS structures [42",43]. These genes have a dramatic effect on the development of neural progenitor cells in particular regions of the nervous

system. Further studies are required to investigate the roles of regionally expressed genes in neural progenitor cells. In the case of BF-1, the authors [42 °] speculate on an influence on neural progenitor cell proliferation.

Neural progenitor cells respond to patterning signals and may themselves perpetuate these signals [44]. However, there is new evidence that progenitor cells may not be committed to a given regional identity, but that they remain responsive to regional signals for some time. When labelled progenitor cells from basal forebrain are re-injected into developing CNS, the cells can integrate into a number of different neural regions, at least in forebrain and midbrain, and develop appropriately [45"',46,47]. The extent of this plasticity remains to be determined.

The neuron/glial fate choice is a fundamental decision point in specifying neural progenitor cells. Genes that may function in denoting a neuronal or glial fate are being explored. Recently, a silencer factor has been identified that turns off a number of neuron-specific vertebrate genes, and it may function in suppressing neuronal fate in ectoderm cells or in making the neuron/glial choice in progenitor cells [48]. Another exciting advance in this area is the cloning of glial cells missing (gcm), a Drosophila gene [49",50"]. This gene encodes a novel nuclear protein that is expressed in nearly all Drosophila CNS and PNS glial cells. In loss-of-function gcm mutants, most glial cells fail to differentiate. Ectopic expression of gcm can drive presumptive neurons to a glial cell fate. Thus, gcm appears to represent a switch that distinguishes between these two fundamental neural cell types, and it will be interesting to test whether a homologue is expressed and has a related function in vertebrates.

In Caenorhabditis elegans, a genc that appears to confer neuronal GABA phenotype has been described recently [51]. Will a similar gene be involved in determination of GABAergic fate in vertebrates? For example, in the cerebral cortex where glutaminergic and GABAergic neurons have separate progenitor cells [7]?

Factors regulating development of neural progenitor cells There has been a recent comprehensive review of factors regulating mammalian neural progenitor cells [2]. There are a number of points that we would like to make in the context of this review.

Advances have been made in the past year in our understanding of exogenous factors that influence pro- liferation and differentiation of neural progenitor cells. Many new reports concern the action of fibroblast growth factors (FGFs) and EGE and it is clear that a single factor can have multiple effects on neural progenitor cells. Basic FGF (bFGF; also called FGF-2) stimulates division of multipotentiai cells from striatum and cerebral

Vertebrate neural progenitor cells Temple and Qian 15

cortex, restricted neuronal progenitor cells from striatum and olfactory epithelium, and restricted glial progenitor cells [2,52,53°]. A recent report suggests that bFGF may also stimulate division of cortical glutaminergic precursor cells, but not GABAergic precursors, which would allow independent regulation of these two major classes of cortical cells [54]. In this study, an increase in glial cells was not found, surprisingly, since bFGF can stimulate division of multipotential cortical cells that generate glial progeny ([2,53°,55]; X Qian, S Temple, unpublished observations), a difference that is probably attributable to culture conditions.

Recently, two neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin (NT)-3, were shown to promote neuronal differentiation from bFGF-treated cortical VZ cells [55,56]. The neurotransmitters glutamate and GABA were both shown to inhibit progenitor cell division in cortical VZ cells, also suggesting a role in progenitor cell differentiation [57°]. In all these cases, the type of progenitor cell influenced remains to be determined.

Two new studies link responsiveness to EGF with gliogenesis. Early cerebral cortex contains a multipotential progenitor cell that is responsive to bFGF but not to EGF [58]. Later, cells responsive to both growth factors arise, and these appear restricted to a glial fate [58]. In retina, overexpression of wild-type EGF receptor stimulates multipotential retinal progenitor cells to make Mtiller glia [59°]. It is not clear whether expression of EGF receptors in cortical progenitors is causally linked to a restriction to glial fate, or is a result of glial restriction stimulated by some other factor(s). In retina, however, the action of EGF appears similar to that of glial growth factor (GGFII; an EGF-related factor) on neural crest stem cells, which is to push multipotential cells to generate glia [60°i.

Timing of differentiation is key to normal neural development: for example, neurons are born on a precise schedule to generate cortical and retinal layers, and, in general, neurons appear before glial cells. Timing may be accomplished by sequential generation of different types of progenitor cells from multipotential cells (Figure 2). In vitro, muitipotential stem cells produce neurons before glial cells ([11°']; X Qian, S Temple, unpublished observations), and in vivo, neuronal progenitor cells are prevalent early in development and glioblasts appear later [12°]. Timing could also occur through independent induction of progenitor cells. In either case, environmental signals probably play a role. For example, differentiated retinal cell produce factors that change the type of neurons produced by retinal progenitor cells [24]. It is important to clarify the cellular and molecular mechanisms underlying timing in neural development. In C. elegans, a hierarchy of

interacting genes regulate timing of developmental events [61], and it will be interesting to investigate whether related genes play a similar role in vertebrates.

Neural progeni tor cells in the adul t In most regions of the mammalian CNS, neurogenesis has ceased by birth. As an exception, neural progenitor cells remain in the subventricular zone (SVZ) into adulthood; however, they have been traditionally seen as glial precursors. Recently, a subpopulation of adult SVZ cells has been shown to generate neurons that migrate into the olfactory bulb [62",63]. In vitro studies suggest that other subpopulations of SVZ cells also retain the capacity to generate neurons. EGF and bFGF can stimulate long-term division of subpopulations of adult murine SVZ cells that can generate neurons and glia [2,53°]. Recent experiments in vivo have provided evidence that the adult rat SVZ also contains a population of slowly dividing stem cells that can replenish a more rapidly dividing SVZ population [64°°]. Are these slow growing stem cells the direct products of embryonic multipotential stem cells that become relatively quiescent in the adult? Or do they represent a cellular compartment that is present throughout development, but has not yet been revealed in that embryo?

The discovery of neural stem cells that can be grown in vitro has raised a great deal of interest because of the possibility of using these cells in replacement therapies. For example, it might be possible to circumvent the problem of host versus graft rejection by utilizing a patient's own neural progenitor cells as donor tissue. In addition, the recent demonstration of plasticity in neural progenitor cells when transplanted to different regions [45"°,46,47] raises the hope that immature neurons generated from cultured stem cells could replace a variety of neuronal types. Currently, few studies have examined what happens when these bFGF- or EGF-expanded progenitor cells are replaced in the CNS [65]. Progress will depend on being able to find conditions under which these cells will generate substantial numbers of mature neurons.

Conclusions Substantial headway is being made in characterizing neural progenitor cells. It is important to clarify the role of multipotential neural progenitor cells in neural development, and to understand how these cells are regulated, especially in the light of their therapeutic promise. We are beginning to identify genes specifying classes of neural progenitor cells, and the factors that regulate them. Intriguing parallels are being uncovered between vertebrate and invertebrate neural progenitor cells, and it will be extremely interesting to see how similar the two systems prove to be.

16 Development

Acknowledgements Thanks to Jeff Stern for reading the manuscript. This work was supported by National Institutes of Health grant RO1 NS 33529-01A1.

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