Neural Development: Fate diverted

KRISTJAN R. JESSEN AND RHONA MIRSKY

NEURAL DEVELOPMENT

Fate diverted Signals that alter cell fate are probably crucial in metazoan development. Glial growth factor may play such a role in the mammalian neural crest, by regulating the generation of neurons and Schwann cells. The body of a multicellular organism is made up of a large number of different cell types. But all of these cells develop from just one cell, the fertilized egg. Two types of cellular event are required for development to occur: proliferation and diversification. Not surprisingly, the regulation of these processes represents a major focus of research in cellular and developmental biology. The idea that proliferation and diversification are intimately linked has gained currency from studies on invertebrate model organisms [1], in which cell divisions that produce two non-identical daughter cells are often observed.

Observations of this type helped give rise to the attractive notion that, during mitosis, a key molecule(s) affecting subsequent cell fate might distribute unevenly between the two daughter cells, maybe by chance. Such a molecule could, for instance, be a cell surface receptor for an instructive environmental signal, such as a polypeptide growth factor. Then, only one of the two daughter cells would be responsive to the instructive signal, which could further change the cell's phenotype, thereby amplifying the initial non-equivalence generated by the asymmetric cell division, and so setting the cell on course down a lineage different from that of its sister. More recent work supports the idea that probabilistic non-equivalences of this type may, in fact, arise quite independently of mitosis (such probabilistic changes are also often referred to as stochastic, autonomous or spontaneous). That is to say, a cell might alter the expression of a lineage-relevant molecule(s) spontaneously, as a consequence of probabilistic intracellular events. An example is provided by two of the cells involved in gonad development in the worm Caenorlhabditis elegans [2].

Fig. 1. Modulation of spontaneous lineage choices.

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These cells, called Zl.ppp and Z4.aaa, are developmentally equivalent - they have identical options for further development: each cell can become either an AC cell or a VU cell. Intriguingly, in normal development, one (and only one) of these cells becomes an AC cell half of the time it is Zl.ppp and half of the time Z4.aaa - while the other cell becomes VU. It has been suggested that this type of situation arises in the following way. If left to its own devices - in the absence of any signal from the environment - each of these cells will change spontaneously to become an AC cell. Each cell also has the potential ability to send a signal to its neighbour that will block this spontaneous change, allowing the neighbouring cell instead to become a VU cell. Which of the two cells becomes the signalling cell is, again, thought to be determined by a spontaneous change, which is unrelated to cell division and which can occur with equal probability in either of the cells. The cell that, by chance, acquires the signalling phenotype will prevent its neighbour from signalling. The signalling cell will, therefore, not receive any blocking signals. As a result, it will spontaneously proceed to become an AC cell. The receptor for the blocking signal might be the product of the lin-12 gene, a member of the lin 12-Notch family that is present in a variety of species.

© Current Biology 1994, Vol 4 No 9

DISPATCH In fact it now seems likely that probabilistic differentiationrelated changes, such as those discussed above, do not have either to be random (all the possible options being adopted with equal frequency) or to occur as a virtual certainty, but can be somewhere in between. This can be inferred from recent experiments on haematopoiesis, the formation of blood cells, in mammals [3]. In these experiments, progenitor cell lines were genetically engineered so that they could survive in culture in the absence of extrinsic growth or signalling factors. It was found that, under these deprived conditions, the progenitor cells nevertheless made various lineage choices, forming several types of mature blood cells, rather than remaining as a homogeneous population of immature cells. From these and related studies emerges a view of cell diversification, in which developing cells spontaneously and probabilistically enter one lineage out of several possible ones, with each choice carrying a certain probability (Fig. la). This is sometimes referred to as the stochastic hypothesis. An alternative, inductive hypothesis says that a cell will only enter a lineage when instructed to do so by an extrinsic signal; according to this model, choice between lineages occurs as a response to distinct extrinsic signals. The stochastic hypothesis raises a fundamental question: can these intrinsically determined probability assignments be altered by extrinsic signals? The answer is almost certainly yes, as shown in Figure la. In fact, interactions between cells and between cells and the extracellular matrix are likely to act as major determinants of developmental decisions. One example of this is seen in the cells of C. elegans discussed above, where signalling alters AC cell fate to VU fate. Systems such as the Zl.ppp and Z4.aaa cells are often encountered in development: a cell, in the absence of extrinsic signals, will invariably choose one fate, and this pathway - that is, the one with a probability of 1.0 - is often referred to as the 'default' pathway (Fig. lb). Another example of a system in which a default pathway has been described is the mammalian 02-A glial progenitor cell in culture: this cell will choose to become an oligodendrocyte in the absence of signals, but can be instructed to become a type II astrocyte by exposure to serum-derived factors [4]. It may also turn out that the much-studied process of neural induction in amphibia, which switches part of the dorsal ectoderm to a neural fate, in fact represents a default pathway, a cellautonomous choice that is prevented from occurring in the surrounding ectoderm by instructive signals [5]. There is no reason to expect to find fundamental differences between systems in which a single default pathway operates and systems in which a cell has more than one spontaneous lineage choice. One test of whether these systems are indeed essentially similar is to ask whether alteration of a spontaneous lineage choice by instructive signals - a key feature of default systems - also occurs in systems in which cells have

Fig. 2. Selective signals alter the composition of populations generated by spontaneous lineage choice. several choices. In other words, can the probabilities that are intrinsically assigned to each of several possible spontaneous lineage choices be modified by extrinsic factors? An affirmative answer is suggested, but not proven, in the haematopoietic study by Fairbairn et al. [3], as they noted quantitative differences in the types of mature cells formed from progenitors depending on the presence or absence of serum. The possibility cannot, however, be excluded that these differences arose after lineage choices were made, and that they are due to differential effects of serum factors on survival or proliferation. Some of these problems have been avoided in a new study of lineage choice carried out on another tissue much favoured for probing these issues, the neural crest [6]. Before discussing the experiments, a very short briefing on this interesting tissue follows [7]. The neural crest is a small and transient population of cells that breaks away from the dorsal aspect of the neural tubeas it closes. The cells soon disperse, migrate away, proliferate and differentiate to form an impressive variety of cell types, including neurons, glial cells, pigment cells, cartilage and bone. It has been shown quite clearly that many neural crest cells are multipotent -

a single neural crest cell can give

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Current Biology 1994, Vol 4 No 9 Fig. 3. Schematic illustration of GGF-II, pro-ARIA and pro-NDFa. The kringle domain, preceded by a signal peptide (SP), is exclusive to the nervous system; an alternative amino-terminal sequence (Alt N Term) is found in non-kringle forms of neuregulin. The glycosylationrich domain (glycos) is more extensive in NDF; and two major variants ( and p) of the receptor-binding EGF-like domain are found. Further variations occur in a sequence distal to the EGF domain, designated 1, 2 or 3, which is a putative site for proteolytic processing. TM, transmembrane; Cyto, cytoplasmic. rise to several types of differentiated derivative. This has been demonstrated both in vivo, by injecting lineage markers into single crest cells and following their subsequent fate, and by clonal culture in vitro. In the latter experiments, individual neural crest cells from chick, quail, mouse or rat are maintained in vitro for a few days, during which they proliferate to form clones of varying sizes. Analyses of such clones using antigenic and morphological criteria generally show that many of them contain a variety of differentiated crest derivatives in various combinations [8,9]. Many investigators have also used mass cultures of neural crest. Although differentiated crest derivatives are readily generated from mass cultures, they offer less scope for analysis. Do extrinsic signalling molecules influence neural crest cell differentiation in these model systems? This question has mostly been studied using mass cultures, but also using clones of cells, and both types of experiment give a similar answer: a variety of identified and unidentified factors can, when added to the culture medium, radically alter the profile of differentiated cells obtained. For example, brain derived neurotrophic factor, leukemia inhibitory factor and retinoic acid each increase the number of neurons or neuronal precursors appearing in crest cultures, whereas Steel factor and at-melanocyte stimulating hormone increase the number of pigment cells observed, and transforming growth factor 01 seems to block pigment cell generation [9,10]. But these observations do not necessarily mean that extrinsic factors affect the lineage choice made by individual crest cells. Two other types of event, selective proliferation and selective survival, can have a striking effect on the outcome of this type of experiment (Fig.2). In this context, signals that affect proliferation or survival are sometimes referred to as selective or enabling signals, to distinguish them from instructive or inductive signals that more directly affect lineage choice. In many systems, including neural crest-derived lineages, clear examples exist of lineage-dependent changes in growth and survival factor requirements of the kind discussed here [11]. Shah and colleagues [6] have attempted to distinguish between instructive and selective signals in their study of the role of glial growth factor (GGF) in neural crest development. GGF has long been suspected to play an

important part in the development of one of the main types of neural crest derivative, Schwann cells, although its precise identity and family relationships are only now emerging, some 25 years after it was first described [12]. GGF when purified, consists of three distinct species, GGF-I (34 kD), GGF-II (59 kD) and GGF-III (45 kD), all of which can stimulate Schwann cell proliferation [13]; the GGFs are alternatively spliced products of a single gene [14]. Surprisingly, multiple independently cloned proteins, including Neu differentiation factor (NDF), heregulins, acetylcholine receptor inducing activity (ARIA) and the GGFs are all products of the same gene (Fig. 3) [15]. The name neuregulin (NRG) has been suggested for members of this protein family [14], but is by no means universally used [15]. NDF and the heregulins were isolated from rat fibroblastic and human mammary cell lines, ARIA from adult chicken brain and the GGFs from bovine pituitary. The diverse origin of these molecules pointed early on to the possibility that neuregulins might have a broad spectrum of activities, rather than being restricted to the nervous system. This idea has been further strengthened by in situ hybridization and immunocytochemical studies, which show that although neuregulins are strongly expressed by neurons (but not by glia) within the developing and adult nervous system [14,16], neuregulin mRNA is also associated with subepithelial mesenchymal tissue during development [16]. Neuregulins are members of the epidermal growth factor superfamily. They bind to tyrosine receptor kinases of the EGF receptor (HER) family; the EGF-like domains of neuregulins contain the region required for receptor activation. Although it was originally thought that NDF bound specifically to, and caused direct phosphorylation of, the HER2 receptor (c-erbB2), recent results using genetically engineered cells expressing only HER2 or a related receptor, HER4 (c-erbB4), or both together, suggest HER4 is the true receptor [17]. Dimerization of HER4 and HER2 to form an active complex, however, probably occurs in some cell systems [15]. Shah et al. [6] have studied how the generation of two cell types, neurons and Schwann cells, is affected in clonal cultures of neural crest cells by the addition of recombinant GGF-II. Under basal growth conditions, about 90 % of the clones surviving after 16 days contain

DISPATCH both neurons and Schwann cells, whereas about 10 % contain Schwann cells only. Addition of GGF alters this outcome radically: less than 5 % of surviving clones now contain both neurons and Schwann cells and over 95 % contain Schwann cells only. The main conclusion drawn by the authors [6] is that GGF is acting as a negative instructive signal, making the choice of a neuronal fate less likely. The major alternative explanation, namely selective effects on proliferation and/or survival, is excluded on the following grounds. Because clonal size at 16 days is similar in the presence and absence of GGF, it is argued that the results could probably not be explained by a selective mitogenic effect of GGF on cells of the Schwann cell lineage. DNA synthesis was not measured directly, and the argument could be countered by invoking a complex combination of mitogenic and survival effects, but this remains unlikely. The possibility that GGF selectively kills cells that enter the neuronal lineage is argued against by the observation that cells expressing MASH1, an early marker of subsets of neuronal progenitors, are not killed by addition of GGF to cultures in which MASH1-positive cells have been allowed to develop. This does not, however, preclude an effect on earlier progenitors - cells that have entered the neuronal lineage but do not yet express MASH1 - nor an effect on neuronal sub-lineages that do not express MASH1, although the authors [6] consider this unlikely. It is also suggested that GGF, in addition to its role in suppressing neuronal differentiation, might have have an instructive role in promoting glial differentiation, although the data appear inconclusive on this point. As this study [6], in agreement with previous work [14,16], shows that GGF is expressed by neurohs and not by neural crest cells or glia, it is proposed that neuronal GGF might act on undifferentiated precursor cells to make the further generation of neurons less likely and the generation of glia more likely. Interestingly, GGF is a homologue of the Lin-3 protein, which controls a cell fate decision regulated in conjunction with the Notch homologue Lin-12 in C. elegans vulval precursor cells. The possibility is therefore raised that mammalian Notch homologues, which are expressed early in peripheral ganglia, could also be involved in neuronal-glial cell fate decisions in the neural crest [18]. It seems likely that, in general, the response of a cell is determined by the total growth factor input, rather than by single factors causing an invariant response regardless of context. GGF seems, in these experiments, to alter the lineage choice of crest cells that already, before addition of GGF, find themselves in a complex cocktail of defined and undefined signalling

molecules (embryo extract). It will be important in the future to define whether the instructive effects of GGF are contingent on any - and if so, which - components of this cocktail. Nevertheless, these experiments come significantly closer than previous studies to demonstrating clearly that an identified growth factor, GGF, can act as an instructive signal and alter lineage choice in neural crest cells.

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Kristjan R. Jessen and Rhona Mirsky, Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK.

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