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Neurogenic genes and vertebrate neurogenesis
Neurogenic genes and vertebrate neurogenesis Julian Lewis The neurogenic genes of the Delta-Notch signalling pathway mediate lateral inhibition - a mechanism that controls cell commitment in many tissues and serves in the developing nervous system to single out cells for a neural fate. Recent work has revealed the outlines of the signal transduction pathway from Notch to the nucleus, has clarified the mechanisms by which lateral inhibition causes adjacent cells to become different, and has shown that vertebrates use essentially the same lateral inhibition machinery as flies and worms to regulate neurogenesis.
Address Imperial Cancer Research Fund (ICRF) Developmental Biology Unit, Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK e-mail: [email protected] Abbreviations AS-C achaete-scutecomplex bHLH basic helix-loop-helix E(spl)-C Enhancer-of-split complex lIES hairy-and-Enhancer-of-split SMC sensorymother cell Su(H) Suppressor of Hair/ess
Current Opinion in Neurobiology 1996, 6:3-10 © Current Biology Ltd ISSN 0959-4388
Introduction A neuron in a fly or a worm is recognizably the same type of cell as a neuron in a vertebrate; but is it generated in the same way? Has evolution conserved not only the cell type, but also the machinery for its production? At first sight, the answer appears to be no: if we compare the developmental anatomy of the CNS of a vertebrate with that of Drosophila, for example, the structures appear radically different. The vertebrate has a dorsally located neural tube, with neurons differentiating within the neuroepithelium, whereas Drosophila has instead a ventrally located chain of segmental ganglia, with neurons generated from neuroblasts that are derived by delamination from the neuroectoderm. Anatomical appearances can be deceptive, however, and in the past few years it has become increasingly clear that remarkably similar sets of genes are involved in neurogenesis in the different classes of animals. Thus, Drosophila and Caenorhabditis elegans offer a key to the understanding of neurogenesis in vertebrates.
This review focuses on one particular subset of the genes that regulate neurogenesis. The members of this subset are called the neurogenicgenes because, in Drosophila, mutations in them result in massive over-production of
neurons. The first part of this review introduces the neurogenic genes through an outline of their role and mechanisms of action in Drosophila neurogenesis, where there have been some important recent advances. Against this background, the second half of the review will discuss new findings on the role of the neurogenic genes in vertebrate neurogenesis. Neurogenic
genes
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Drosophila neurogenesis Work over many years in flies and worms [1-3,4",5"] has identified the products of the neurogenic genes as the components of a cell-cell signalling mechanism whereby, in the nervous system, a cell that becomes committed to a neural fate inhibits its neighbours from doing likewise. In this process of lateral inhibition, the receptor for the inhibitory signal is a transmembrane protein encoded by a neurogenic gene called Notch in Drosophila, and the ligand, expressed in the cell delivering inhibition, is another transmembrane protein encoded by a neurogenic gene called Delta in Drosophila. The inhibitory signals are exchanged within groups of cells, so-called proneural clusters, that all have the potential for a neural fate; the role of lateral inhibition is to ensure that only a scattered subset of the cells actually become committed to that fate. The proneural clusters are defined by expression of proneural genes, chiefly the members of the achaete-scute complex (AS-C). The products of the AS-C genes are basic helix-loop-helix (bHLH) transcription factors, which have an initial upstream role as inducers of the expression of neurogenic genes throughout the proneural cluster. T h e y also have a subsequent downstream role, in that their own expression is, in turn, regulated by the lateral inhibition machinery: as cells become committed to a neural fate, their content of AS-C proteins increases, whereas that of their neighbours in the cluster decreases [6]. The heightened expression of AS-C genes in the nascent neural cell is transient, as is the expression of Delta [7,8]. In the embryonic CNS of Drosophila, neuroblasts are generated from the neuroectoderm in successive waves [9], with the proneural and neurogenic genes controlling the proportion of cells that are singled out for this fate in each wave of neurogenesis. Each neuroblast will then undergo repeated asymmetric divisions to generate neurons and/or glial cells (see review by Doe and Skeath, this issue, pp 18-24). In the PNS, the same genes control the singling-out of cells from the epidermis to be sensory mother cells (SMCs; also called sense-organ precursors, or SOPs), each of which will undergo two or more asymmetric divisions to generate the neuronal and supporting cells of a single bristle or other sensillum [2]. In this latter
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process the neurogenic genes are required a second time, providing lateral inhibition to prevent all the progeny of the precursor from becoming neurons [10]. Outside the nervous system, the same genes govern cell differentiation in tissues as varied as the gut epithelium [11"], the musculature [12], and the Malpighian tubules [13]. In each case, the same lateral inhibition mechanism appears to operate, generating a fine-grained pattern in which individual cells are singled out for commitment to a particular mode of differentiation, while the adjacent cells are inhibited from entering upon that commitment.
Recent work has demonstrated at least one pathway by which lateral inhibition with feedback can come about (Figure 1). Activation of Notch induces expression of genes of the Enhancer-of-split complex (E(spl)-C) [18,19"]; these code for repressive b H L H proteins that act, in conjunction with the co-repressor Groucho [20,21"], to downregulate expression of genes of the AS-C [22°]; the AS-C genes, in turn, code for activator b H L H proteins that promote expression of Delta [23]. The net effect is that Notch activation reduces Delta expression by reducing AS-C expression [22"].
How to make neighbours different
Asymmetric cell division The other mechanism for generating asymmetric cell fates during Drosophila neurogenesis depends on asymmetric cell division: two sister cells adopt different fates because they inherit different quantities of a regulatory protein (or set of proteins) that becomes concentrated at one pole of the parent cell. As reviewed elsewhere ([24"]; see also the review by Doe and Skeath, this issue, pp 18-24), at least two proteins, Numb and Prospero, behave in this way both during the asymmetric divisions of neuroblasts in the CNS and during those of SMCs in the PNS [25-29]. These proteins are necessary and sufficient to confer a neural fate on the daughter that inherits them.
In all the examples mentioned above, and in comparable processes in vertebrate neural development, neighbouring cells have contrasting fates. What makes them behave differently? Why, for example, don't all the cells in a proneural cluster remain locked in a state of mutual inhibition, each cell both delivering inhibition to its neighbours and receiving inhibition from them? Two mechanisms have been proposed, with good evidence in Drosophila for both. They are not mutually exclusive, and both must be borne in mind when considering how neighbouring cells come to adopt different fates during vertebrate neurogenesis. Lateral inhibition with feedback The first mechanism can be called lateral inhibition with feedback. It rests on an elaboration of the basic lateral inhibition mechanism, such that the more inhibition a cell receives, the less it is able to deliver [3,14,15]. In molecular terms, this means that increased activation of Notch in a given cell causes a decrease in the amount or activity of Delta in that cell. In a system of two or more cells, this creates a positive feedback loop (Figure 1), because the cell that delivers more inhibition to its neighbours drives down the level of inhibition that it receives back from them, and thereby still further increases its own ability to deliver inhibition. Thus, any initial a s y m m e t r y - - e v e n a small o n e - - b e t w e e n two adjacent cells will be self-magnifying, leading to a state where one cell predominantly receives inhibition and does not deliver it, while the other predominantly delivers it and does not receive it.
Persuasive evidence for this mechanism has come from studies of the way in which cells are singled out to become SMCs in the developing PNS. For example, at the border of a mutant clone where cells with different numbers of copies of the Delta gene confront one another, it is the cells with more Delta gene copies that become SMCs, whereas their neighbours with less copies are inhibited [15]. Strong evidence for lateral inhibition with feedback has come also from work in C. elegans, where the Notch homologue lin-12 and the Delta homologue lag-2 act in this way to control the genesis of differences between initially equivalent cells in the gonad and elsewhere [3,16",17].
In the case of the SMC, there seems at first to be a paradox, in that Delta-Notch signalling is also required to ensure that the daughters of the SMC become different and do not both become neural [10]. It is not hard to see, however, how lateral inhibition may operate in concert with neural determinants such as Numb and Prospero. Suppose, for example, that in the daughters of the SMC, lateral inhibition operates without feedback, so that the daughters persist in delivering inhibition to one another symmetrically. If the neural determinants are absent, both daughters will remain non-neural; if one daughter inherits the neural determinants, and these make her immune to the inhibitory signal, she alone will become neural; and if Delta-Notch signalling is inoperative, neither daughter will be inhibited, and both will become neural; all this fits the facts. There is indeed evidence suggesting that lateral inhibition between the progeny of the SMC does operate without feedback: the AS-C gene products disappear from the SMC before its first differentiative division and play no part in controlling the fate of its progeny [7], so there cannot be any feedback of Notch on Delta via AS-C genes in this case.
From membrane to nucleus: the mechanism of signal transduction by Notch Regardless of whether lateral inhibition operates with feedback or without, Delta-Notch signalling poses the perennial problem of signal transduction: how does activation of Notch at the cell membrane bring about changes in the pattern of gene expression? Several recent studies, in flies, worms, and vertebrates, together make a strong case for a novel and unusually direct mechanism
Neurogenic genes and vertebrate neurogenesis Lewis
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Figure 1
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© 1996 Current Opinion in Neurobiology
The Delta-Notch pathway for lateral inhibition according to the current view, depicted for a pair of adjacent proneural cells. Only the components discussed in this review are shown; although the signalling pathway is known to depend on several additional components, their specific roles are still obscure. DNA-binding proteins that function as repressors are marked with a '-', those that function as activators with a '+'. A dashed arrow symbolizes a regulatory link between AS-C genes and Delta that provides for feedback in the lateral inhibition of one cell by the other; Ac/Sc stands for Achaete/Scute proteins - the products of the AS-C. The regulatory links shown probably do not represent the only means by which Notch activation exerts its effects. Some evidence suggests, for example, that the main control of Delta activity during the singling-out of neural cells in Drosophila is post-translational rather than transcriptional [8].
(Figure 1): ligand binding causes cleavage of the Notch protein, releasing from it an intracellular fragment, called NotchlC, which then moves into the nucleus to regulate transcription [30",31",32]. Experiments in mammalian cells show that mutant forms of Notch lacking most of the extracellular domain but retaining the transmembrane domain undergo cleavage to release NotchlC, even in the absence of ligand binding [31"]. In transgenic fly and worm embryos, and in cultured mammalian P19 cells, both NotchlC and these other truncated forms of Notch have a dominant antineurogenic effect, inhibiting neural differentiation in the manner expected if there is constitutive activation of the Notch signalling pathway [32-37]. NotchlC has nuclear localization signals and includes a domain that mediates binding to the product of another neurogenic gene, Suppressor of Hairless (Su(H)) [38",39°]. In Drosophila $2 cultured cells, activation of Notch by Delta leads to a movement of Su(H) protein into the nucleus [38"']. In the nucleus, Su(H) acts by binding to regulatory regions of the E(spl)-C and inducing transcription of E(spl)-C genes [40"], and studies of the homologous genes in mammalian cells indicate that this action of Su(H) depends on the presence of NotchlC bound to it [30"'].
'Activated Notch' constructs--that is, NotchlC and other forms of Notch lacking the extracellular d o m a i n - - a r e powerful tools for the analysis of Notch-dependent signalling mechanisms. For example, they have been used in combination with loss-of-function mutations of other neurogenic genes to discover the epistatic ordering of these genes; these tests confirm that the E(spl)-C lies downstream from Notch, that Delta is upstream from Notch, and that the neurogenic genes mastermind (mare), big brain (bib), neuralized (neu) and almondex (amx) also lie upstream from Notch [ 3 2 ] - - t h a t is, the presence of activated Notch causes an antineurogenic phenotype regardless of whether these genes are functional. T h e precise roles of mam, bib, neu and amx remain a mystery, however.
Neurogenic genes in vertebrates In vertebrates, there have been only two reports of neurogenic genes discovered by the methods of classical genetics: one is called white tail and was found in the Ttibingen zebrafish mutagenesis screen [41"], and the other is called mind bomb and was found in the Boston zebrafish screen [42°]. It is possible that they both are alleles of the same gene. By comparison, the number of vertebrate genes identified by homology with Drosophila
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neurogenic genes is large. First to be discovered was
Xotch, a Xenopus Notch homologue, now renamed X-Notch-1 [43]. Since then, multiple Notch homologues have been found in rat, mouse, human, zebrafish and chick (reviewed in [5°]). More recently, homologues of Delta have been found [44",45",46",47], as well as homologues of the Delta-related gene Serrate [48"',49"]. There is a whole family of homologues of E(spl)-C b H L H genes [50"',51], known as HES g e n e s - - f o r hairy-and-Enhancer-of-split h o m o l o g u e s - - b e c a u s e they also resemble the Drosophila gene hairy. There is also a family of vertebrate groucho homologues, called TLE genes [52,53]. Su(H) turns out to have as its homologue a vertebrate gene previously identified in another context as coding for a transcription factor variously called RBP-J~ CBF1 or KBF2 [39",54,55]. T h e vertebrate achaete-scute homologues (ASH genes) are again numerous; their role in vertebrate neurogenesis is reviewed elsewhere [56"], and is a large topic that will not be covered here except in passing.
Vertebrate Notch and Delta homologues and lateral inhibition in primary neurogenesis In vertebrates, neurons are generated in at least three distinct ways: within the central neuroepithelium that forms the neural plate and neural tube; by migration from the neural crest; and by delamination from the cranial placodes. Although placodal neurogenesis shows the most intriguingly close anatomical parallels with insect neurogenesis [57], I shall concentrate here on central neurogenesis, on which most of the recent relevant research has been done. In the central neuroepithelium, the first neurons appear as scattered postmitotic cells in a number of distinct regions (comparable with proneural clusters in the insect neuroectoderm), where they are mingled with undifferentiated proliferative neuroepithelial cells. T h e pattern is most strikingly displayed in the neural plate of amphibians and fish, where production of a set of primary neurons is separated by a time-lag from production of subsequent sets of neurons [58,59]. Notch-l, and possibly other Notch homologues, are expressed by all the cells in the regions where primary neurogenesis is occurring. Meanwhile, expression of the Delta homologue Delta-1 is r e s t r i c t e d - in Xenopus and chick, at l e a s t - - t o a scattered subset of cells that have ceased dividing and appear to be the nascent neurons [45",46"]. This suggests that Delta-Notch signalling is operating as in the fly to single out cells for a neuronal fate. Gene functions can be tested directly in Xenopus by injecting RNA into the embryo at the two-cell stage. When RNA coding for full-length Deha-1 protein is injected, the production of neurons is inhibited [45°']: forced expression in all cells of a gene normally expressed only in nascent neurons inhibits all cells from becoming nascent neurons. This behaviour, at first sight paradoxical, is the hallmark of a gene whose function is to deliver
lateral inhibition: normally, isolated cells express the gene and inhibit their neighbours, from whom they receive no inhibition; but when all cells are forced to express the gene, they all inhibit one another. Injection of RNA coding for a modified Delta-1 protein with a truncated intracellular domain has a contrary effect, causing excess production of neurons, as though it is blocking the normal delivery of inhibition by the endogenous Deha-1. Injection of RNA coding for activated N o t c h - - e i t h e r NotchlC, or Notch-1 with a truncated extracellular d o m a i n - - n o t only prevents the production of neurons, as expected if NotchlC inhibits commitment to a neural fate, but also inhibits expression of the endogenous Delta-1 gene [45"°]. This implies the existence of a feedback in the lateral inhibition system, of the type described above for Drosophila (see Figure 1): a cell expressingDelta-1 more strongly than its neighbours will not only tend to inhibit them from becoming neurons, but will also inhibit them from delivering inhibition reciprocally and so will reinforce its own advantage. In this way, starting perhaps with uniform low-level expression of Delta-1 or with uniform expression of some other Delta homologue, cells within an initially homogeneous population may become singled out for a neural fate.
Vertebrate Notch and Delta homologues in later neurogenesis Is this conserved machinery of lateral inhibition with feedback a peculiarity of primary neurogenesis, or does it operate in subsequent neurogenesis too? After the first neurons in a vertebrate have been generated, the undifferentiated neuroepithelial cells of the embryonic CNS serve as stem cells for the production of further neurons. Over a period of many days, they continue to divide, with a proportion of their progeny remaining proliferative and a proportion withdrawing from the cell cycle and embarking on differentiation as neurons [60,61]. A layered structure soon develops, with the cell bodies of the proliferative cells confined to the ventricular zone (at least in hindbrain and spinal cord), and the postmitotic nascent neurons moving out of this region into the mantle zone as they begin to differentiate. Studies in the chick show that Notch-1 meanwhile is expressed in apparently all cells in the ventricular zone, while Delta-1 is expressed in a non-dividing subset of cells that are scattered within the ventricular zone and appear, once again, to be nascent neurons [49"]. Expression of both genes becomes switched off by the time the nascent neuron moves from the ventricular zone to the mantle zone. All this amounts to strong circumstantial evidence for continuing control of neurogenesis by the same Delta-Notch signalling mechanism as in Drosophila. More direct proof comes from experiments on the developing chick retina [62"']. Activated Notch, carried into the cells by a retroviral vector, inhibits production of neurons; conversely, antisense oligonucleotides targeted against Notch-1 cause
Neurogenic genes and vertebrate neurogenesis Lewis
increased production of neurons. And when the retinal cells are co-cultured with Drosophila cells expressing Delta, they are inhibited from giving rise to neurons. Similarly, in the Xenopus retina, activated Notch inhibits neuron production [63]. T h e neuroepithelium of the developing vertebrate CNS can thus be compared with the neuroectoderm of Drosophila, and the nascent neurons of the one with the neuroblasts of the other. In both cases, scattered cells within a neuroepithelium become singled out for a neural fate, transiently express high levels of Delta, and segregate from their close neighbours expressing Notch, which are inhibited from differentiating and remain to generate subsequent batches of neural cells. There are, however, some important differences: in the vertebrate CNS, the nascent neurons have stopped dividing, whereas in Drosophila, the neuroblasts continue to divide; and in the vertebrate CNS, the nascent neurons stay within the neuroepithelium, whereas in Drosophila, the neuroblasts escape from it. Vertebrate placodal neurogenesis is more similar to insect neurogenesis in these respects, and indeed Notch and Delta homologues are expressed in placodal epithelium at sites of neurogenesis (J Adam, D Henrique, A Myat, unpublished data). It is not clear what part, if any, the Delta-Notch signalling mechanism plays in creating the differences among neuronal and glial cell types in the vertebrate CNS, and there is reason to suspect that asymmetric cell divisions, as well as lateral inhibition with feedback, are important. In the developing cerebral cortex of a mammal, cells in the proliferative zone are seen to divide symmetrically when generating pairs of daughters that both remain in the proliferative zone, but asymmetrically and with a differently oriented mitotic spindle when one of the daughters is destined to emigrate from the proliferative zone (presumably to become a neuron) [64"']. In the latter case, the asymmetry is marked chemically by the distribution of Notch-1 protein; puzzlingly, this becomes concentrated at the basal pole of the dividing cell and is segregated to the emigrating (neuronal) daughter (as one might expect for N u m b and Prospero proteins, rather than Notch, in corresponding circumstances in an insect).
Alternative ligands for Notch From the examples studied so far, it seems that DeltaNotch signalling has a universal role in the control of vertebrate neurogenesis. There is, however, at least one important qualification: Drosophila possesses another gene, Serrate, that also codes for a Notch ligand, and it seems that homologues of Serrate also have a role as Notch ligands during vertebrate neurogenesis in certain regions of the nervous system. Serrate is structurally related to Delta and contains a similar highly conserved Notch-binding domain (the DSL domain [65]), but it acts in different circumstances--for example, in Drosophila, it does not control neurogenesis, but controls the creation of the wing
7
margin, a specialized band of tissue with a key organizing role in the growth of the wing imaginal disc [66-68]. In artificial conditions, Serrate can nevertheless serve to regulate neurogenesis [69]. In the rat, a homologue of Serrate, called Jagged, is expressed in the CNS in restricted domains of the ventricular zone, and, in an in vitro assay, is capable of activating Notch-1 to control gene expression [48"']. Studies of the corresponding chick gene, called C-Serrate-I, show that it is expressed in the ventricular zone of the neural tube in narrow stripes that extend from the anterior part of the hindbrain all the way down the spinal cord [49"]. These stripes correspond exactly to gaps in the expression domain of C-Delta-l, and the cellular pattern of C-Serrate-1 expression within them suggests that C-Serrate-1 may here act instead of C-Delta-1 as a Notch ligand. T h e analogy with the fly, however, would suggest that C-Serrate-1 is more than just a stand-in for C-Delta-l, and that something special is happening at these sites. A still richer vein of speculation is opened up if, as one school of thought has argued, Notch proteins act as receptors not only for members of the Delta/Serrate family, but also as receptors for the signalling protein Wingless and its vertebrate relatives, the Wnts [66,70].
Vertebrate E(spl) homologues: lessons from transgenic mice Notch and Delta are not the only neurogenic genes for which there is direct evidence of a conserved function in neurogenesis. T h e mouse hairy-E(spl) homologue HES-1, for example, is also expressed throughout the ventricular zone, and its role there has been tested both by overexpressing the gene using a retroviral vector and by knocking it out by homologous recombination [50",51]. In accordance with the function of E(spl)-C genes in Drosophila, cells forced to express HES-1 persistently are inhibited from adopting a neuronal or glial fate and remain in the ventricular population, eventually becoming ependymal cells or dying. Conversely, in the HES-I knockout animal, some classes of neurons are generated prematurely. These effects, again as expected by analogy with the fly, correlate with changes in the expression of AS-C homologues. Knockouts of Notch-I and RBP-JK/Su(H) have been less informative, perhaps because of genetic redundancy [71,72]: in both cases, the mutant embryos die half way through gestation, and the relatively mild abnormalities seen in the cytoarchitecture of the neural tube at this stage are difficult to decipher [55,73,74].
Conclusions Lateral inhibition mediated by Delta-Notch signalling appears as a key mechanism in the control of neurogenesis. It seems to be a general principle that neurons are generated, and subsequently function, within a matrix of cells of other types; lateral inhibition provides the means to single them out from their neighbours for their special fate. T h e recent discoveries have revealed in outline how the signalling pathway works, and have shown, for several
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of the main components, that it is similar in vertebrates, flies and worms. T h e course of evolution has indeed conserved not only the neuron as a specialized cell type, but also a major part of the developmental mechanism that gives rise to it. T h e task now is to pursue the many components of the Delta-Notch signalling pathway that remain mysterious (e.g. see [75"]), to find out more about how this mechanism of lateral inhibition is combined with other developmental controls, and to define precisely the cell-fate decisions that it governs, not only in the nervous system but also outside it.
Acknowledgements I thank D lsh-Horowicz for comments on the manuscript, the members of his lab and my lab for many discussions, and P Simpson, I: Guillemot, S Bray, R Kopan, F Schweisguth, Y-J Jiang, A Schier, C Cepko, 1 Greenwald, W Driever and A Martinez-Arias for copies of their papers in advance of publication. I am indebted to the Imperial Cancer Research Fund for support.
References and recommended reading "Papers of particular interest, published within the annual period of review, have been highlighted as: • •.
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Steinberg PW: Falling off the knife edge. Curt Bio/1993, 3:763-765.
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11. •
TepassU, Hartenstein V: Neurogenic and proneural genes control cell fate specification in the Drosophila endoderm. Development 1995, 121:393-405. Shows that the whole system of neurogenic and proneural genes operates in almost the same way in the gut as in the nervous system to single out a subset of cells for a special fate. 12.
Carmena A, Bate M, Jimenez F: lethal of scute, a proneural gene, participates in the specification of muscle progenitors during Drosophila embryogenesis. Genes Dev 1995, 9:2373-2383.
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Hoch M, Broadie K, Jackle H, Skaer H: Sequential fates in a single cell are established by the neurogenic cascade in
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Goriely A, Dumont N, Dambly-Chaudi~re C, Ghysen A: The detarmlnaUon of sense organs in Drosophila: effect of neurogenic mutations in the embryo. Development 1991, 113:1395-1404.
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Heitzler P, Simpson P: The choice of cell fate in the epidermis of Drosophila. Cell 1991, 64:1083-1092.
16. •
Wilkinson HA, Fitzgerald K, Greenwald I: Reciprocal changes in expression of the receptor lin-12 and its ligand lag-2 prior to commitment in a C. elegans cell fate decision. Ce//1994, 79:1187-1198. A thoroughly analyzed example of lateral inhibition with feedback, which serves to single out one cell of an initially equivalent pair to become the anchor cell of the nematode gonad. In this system, activation of the Notch homologue/in-12 causes downregulation of expression of the Delta homoIogue lag-2, as well as stimulating expression of lin-12 itself. 17.
Henderson ST, Gao D, Lambie EJ, Kimble J: lag-2 may encode a signaling ligand for the GLP-1 and LIN-12 receptors of C. elegans. Development 1994, 120:2913-2924.
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JenningsB, Preiss A, Delidakis C, Bray S: The Notch signalling pathway is required for Enhancer of split bHLH protein expression during neurogenesis in the Drosophila embryo. Development 1994, 120:3537-3548.
JenningsB, De Cells J, Delidakis C, Bray S: Role of Notch and achaete/scute complex in the expression of Enhancer of split bHLH proteins. Development 1995, 121:3745-3752. Expression of E(spl)-C genes, assayed with antibodies against the protein products, is shown to correlate with the level of Notch activation in the same cell, both in the embryonic CNS and in imaginal discs. Ectopic expression of an activated Notch construct causes ectopic accumulation of E(spI)-C proteins, even in the absence of AS-C proteins. Thus, E(spl)-C genes are directly regulated by Notch, rather than by the products of the proneural genes. 19. •
20.
Schrons H, Knust E, Campos-Ortega JA: The Enhancer of split complex and adjacent genes in the 96F region of Drosophila melanogaster are required for segregation of neural and epidermal progenitor cells. Genetics 1992, 132:481-503.
21. •
ParoushZ, Finley RL, Kidd T, Wainwright M, Ingham PW, Brent R, Ish-Horowicz D: Groucho is required for Drosophila neurogenesis, segmentation, and sex determination and interacts directly with hairy-related bHLH proteins. Cell 1994, 79:805-815. Clarifies the role of groucho as a neurogenic gene and its relationship to the function of E(spl)-C. Groucho protein binds directly to a subset of bHLH proteins, including those encoded by E(spl)-C. These Groucho partners all function as repressors of transcription and require Groucho as co-repressor. 22. •
Heitzler P, Bourouis M, Ruel L, Carteret C, Simpson P: Genes of the Enhancer of split and achaete-scute complexes are required for a regulatory loop between Notch and Delta during lateral signalling in Drosophila. Development 1996, 122:161-171. By analysis of genetic mosaics in the Drosophila PNS containing clones of cells that lack E(spl)-C genes or groucho, it is shown that the products of these genes are required for reception of inhibition but not for delivery of inhibition (i.e. production of Delta), and that they act downstream of Notch and upstream of the AS-C genes whose transcription they directly inhibit. Clones lacking AS-C genes as well as E(spl)-C genes fail to make sensory bristles and do not deliver inhibition to their neighbours (i.e. do not produce Delta). Thus, AS-C genes are apparently required for production of Delta, and Notch activation inhibits Delta production via E(spl)-C-dependent inhibition of the AS-C genes. 23.
KunischM, Haenlin M, Campos-Ortega JA: Lateral inhibition mediated by the Drosophila neurogenic gene Delta is enhanced by proneural proteins. Proc Nat/Acad Sci USA 1994, 91:10139-10143.
24. •e
Doe CQ, Spana EP: A collection of cortical crescents: asymmetric protein localization in CNS precursor cells. Neuron 1995, 15:991-995. Excellent brief summary of the recent evidence that asymmetric localization of Numb and Prospero at one pole of a dividing neuroblast or sensory mother cell controls the difference of fate between the two daughter cells. 25.
Knoblich JA, Jan LY, Jan YN: Asymmetric segregation of Numb and Prospero during cell division. Nature 1995, 377:624-627.
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RhyuMS, Jan LY, Jan YN: Asymmetric distribution of numb protein during division of the sensory organ precursor cell confers distinct fates to daughter cells. Cell 1994, 76:477-491.
Neurogenic genes and vertebrate neurogenesis Lewis
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Hirata J, Nakagoshi H, Nabeshima Y, Matsuzaki F: Asymmetric segregation of the home•domain protein Prospero during Drosophila development. Nature 1995, 377:627-630.
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Spana EP, Kopczynski C, Goodman CS, Doe CQ: Asymmetric localization of numb autonomously determines sibling neuron identity in the Drosophila CNS. Development t 995, 121:3489-3494.
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Spana EP, Doe CQ: The prospero transcdption factor is asymmetrically localized to the call cortex during neuroblast mitosis in Drosophila. Development 1995, 121:3187-3195.
30. • ,,
JarriaultS, Brou C, Log•at F, Schroeter EH, Kopan R, Israel A: Signalling downstream of activated mammalian Notch. Nature 1995, 377:355-358. Experiments with transfected mammalian cells in culture show that an intracellular Notch fragment binds to the Su(H) homologue RBP-JK, and that the two proteins together form a complex in the nucleus that binds to promoter regions of the E(spl) homologue HES-1, and presumably activates transcription. 31. •o
Kopan R, Schroeter EH, Nye JS, Weintraub H: Signal transducUon by activated mNotch: importance of proteolytic processing and its regulation by the extracellular domain. Proc Nat/Acad Sci USA 1996, in press. Cultured mammalian cells transfected with various mouse Notch constructs are used to show that Notch activation depends on cleavage at a site just internal to the transmembrane domain, producing a NotchlC fragment that moves into the nucleus. The cleavage occurs constitutively, giving an activated Notch phenotype, when the protein lacks a specific part of its extracellular domain. The normal intact extraoellular domain presumably regulates cleavage in response to ligand binding. 32.
Lieber T, Kidd S, Alcamo E, Corbin V, Young MW: Antineurogenic phenotypes induced by truncated Notch proteins indicate e role in signal transduction and may point to s novel function for Notch in nuclei. Genes Dev 1993, 7:1949-1965.
where a normal embryo would generate a single isolated Mauthner neuron, white tail has a cluster of three or four such neurons. There are defects also in the development of somites and in the production of melanocytes (hence the name), and the embryo dies before it is two days old. It remains to be seen whether white tail corresponds to any of the neurogenic genes of Drosophila. 42. •
Schier AF, Neuhauss SCF, Harvey M, Malicki J, Solnica-Krezel L, Stainier DYR, Zwartkruis F, Abdelilah S, Stemple DL, Rangini Z et aL: Mutations affecting the development of the embryonic zebrafish brain. Development 1996, in press. The mind bomb gene has a neurogenic mutant phenotype similar to that of white tail (see [41 "]). Two alleles have been found. 43.
Bettenhausen B, Hrabe de Angelis M, Simon D, Guenet J-L, Gossler A: Transient and restricted expression during mouse embryogenesis of DIll, a murine gene closely related to Drosophila Delta. Development 1995, 121:2407-2418. Describes the cloning and expression pattern of a mouse Delta homologue, Dill. Expression is seen in various non-neural tissues, as wall as in the nervous system. 45. ••
Chitnis A, Henrique D, Lewis J, Ish-Horowicz D, Kintner C: Primary neurogenesis in Xenopus embryos regulated by a homologue of the Drosophila neurogenic gene Delta. Nature 1995, 375:761-766. A Xenopus homologue of Delta is transiently expressed in nascent primary neurons. Its function is tested by injection of RNA constructs into the twocell embryo. The results imply that homologues of Notch and Delta control primary neurogenesis in Xenopus by mediating lateral inhibition, whereby each nascent primary neuron prevents its neighbours from becoming primary neurons. 46. •
Henrique D, Adam J, Myat A, Chitnis A, Lewis J, Ish-Horowicz D: Expression of a Delta homologue in prospective neurons in the chick. Nature 1995, 375:787-790. A chick homologue of Delta is described, and the cells in the early CNS that express it are identified as nascent neurons by virtue of their non-proliferative state.
Rebay I, Fehon RG, Artavanis-Tsakonas S: Specific truncations of Drosophila Notch define dominant activated and dominant negative forms of the receptor. Cell 1993, 74:319-329.
34.
Struhl G, Fitzgerald K, Greenwald I: Intrinsic activity of the Lin-12 and Notch intracellular domains in vivo. Cell 1993, 74:331-345.
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35.
Fortini ME, Rebay I, Caron LA, Artavanis-TsakonasS: An activated Notch receptor blocks cell fate commitment in the developing Drosophila eye. Nature 1993, 365:555-55?.
48. ••
36.
Roehl H, Kimbte J: Control of cell fate in C. elegans by a GLP-1 peptide consisting primarily of enkyrin repeats. Nature 1993, 364:632-635.
3?.
Nye JS, Kopan R, Axel R: An activated Notch suppresses neurogenesis and my•genesis but not gliogenesis in mammalian cells. Development 1994, 120:2421-2430.
Fortini ME, Artavanis-Tsakonas S: The Suppressor of Hairless protein participates in Notch receptor signalling. Cell 1994, 79:273-282. Notch is shown to bind to Su(H), and Notch activation by Delta in Drosophila cultured $2 cells is shown to lead to a translocation of Su(H) into the nucleus. 39. •
Tamura K, Taniguchi Y, Minoguchi S, Sakai T, Tun T, Furukawa T, Honjo T: Physical Interaction between a novel domain of the receptor Notch and the transcription factor RBP-JK/Su(H). Curr Bio11995, 5:1416-1423. Using the yeast two-hybrid system and direct binding assays, the authors show that Notch proteins bind to RBP-JK/Su(H) via a short (<50 amino acids) domain of Notch just internal to the transmembrane domain. This domain does not include the CDC10/ankyrin repeats, previously thought to mediate the binding. 40.
Lecourtois M, Schweisguth F: The neurogenic Suppressor of Hairless DNA-binding protein mediates the transcriptional activation of the Enhancer of split Complex genes triggered by Notch. Genes Dev 1996, in press. Mutant embryos deprived of maternal as well as zygotic Su(H) show a fullblown neurogenic phenotype and fail to express genes of the E(spl)-C. Su(H) protein binds to regulatory sequences of two genes (m5 and me) of the E(spl)-C and is required for the activation of their transcription. •
41. ••
Jiang Y-J, Brand M, Heisenberg C-P, Beuchle D, Furutani-Seiki M, Kelsh RN, Warga RM, Granato M, Haffter P, Hammerschmidt M e t a/.: Mutations affecting neurogenesis and brain morphology in the zebrafish, Danio rerio. Development 1996, in press. white tail mutant embryos produce primary neurons in excessive numbers, but in a normal distribution, just as though there has been a failure of lateral inhibition within proneural clusters; for example, at the site in the hindbrain
Coffman C, Harris W, Kintner C: Xotch, the Xenopus homolog of Drosophila Notch. Science 1990, 249:1438-1441.
44. •
33.
38. ,,•
9
Nye JS, Kopan R: Vertebrate ligands for Notch. Curr Bio11995, 5:966-969.
Lindsell CE, Shawber CJ, Boulter J, Weinmaster G: Jagged: a mammalian ligand that activates Notch1. Cell 1995, 80:909-917. Jagged is a mammalian homologue of the Delta-related gene Serrate, and is expressed in the developing CNS. Experiments with transfected cells in culture demonstrate that, like Delta and Serrate, Jagged can serve as an activating ligand for the Notch signalling pathway. 49. •
Myat A, Henrique D, Ish-Horowicz D, Lewis J: A chick homologue of Serrate, and its relationship with Notch and Delta homologues during central neurogenesis. Dev Bio/1996, in press. Chick homologues of Notch, Delta and Serrate are expressed in the ventricular zone of the neural tube, at late as well as early stages, in a manner suggesting a continuing role in the control of neurogenesis by lateral inhibition. In hindbrain and spinal cord, a curious complementarity is seen between the expression domains of the Serrate and Della homologues, hinting that they may have partly interchangeable functions. 50. ••
IshibashiM, Moriyoshi K, Sasai Y, Shiota K, Nakanishi S, Kageyama R: Persistent expression of helix-loop-helix factor HES-1 prevents mammalian neural differentiation in the central nervous system. EMBO J 1994, 13:1799-1805. A retrovirus is used to force expression of the E(spl)-C homologue HES-1 in cells in the ventricular zone of the embryonic mouse CNS. The infected cells, in contrast to controls, fail to leave the ventricular zone and fail to turn into neurons or gila. Neural precursor cells similarly infected in culture fail to differentiate as neurons. Thus, HES-1, like E(spl), inhibits commitment to a neural fate. 51.
IshibashiM, Ang S-L, Shiota K, Nakanishi S, Kageyama R, Guillemot F: Targeted disruption of mammalian hairy and Enhancer of split homologue-1 (HES-1) leads to up-regulation of neural helix-loop-helix factors, premature neurogenesis and severe neural tube defects. Genes Dev 1995, 9:3136-3148.
52.
Stifani S, BlaumueUer CM, Redhead NJ, Hill RE, ArtavanisTsakonas S: Human horn•logs of a Drosophila Enhancer of Split gene product define a novel family of nuclear proteins. Nature Genet 1992, 2:119-127.
53.
Dehni G, Liu Y, Husain J, Stifani S: TLE expression correlates with mouse embryonic segmentation, neurogenesis, and epithelial determination. Mech Day 1995, 53:369-381.
10
Development
54.
Schweisguth F, Posakony JW: Suppressor of Hairless, the Drosophila homolog of the mouse recombination signalbinding protein gene, controls sensory organ cell fates. Cell 1992, 69:1199-1212.
Time-lapse observation of slice preparations of ferret cortex show that cells emigrating from the proliferative zone are produced by asymmetric cell divisigns. Notch-1 protein appears to be segregated into the emigrant daughter cell, although it is not clear what function, if any, the protein has there.
55.
Oka C, Nakano T, Wakeham A, De la Pompa JL, Mori C, Sakai 1, Okazaki S, Kawalchi M, Shiota K, Mak TW, Honio T: Disruption of the RBP-JK gene results in early embryonic death. Development 1995, 121:3291-3301.
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Tax FE, Yeargers JJ, Thomas JH: Sequence of C elegens leg-2 reveals a cell-signalling domain shared with Delta and Serrate of Drosophila. Nature 1994, 368:150-154.
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Couse JP, Knust E, Martinez Arias A: Serrate and wingless cooperate to induce vestigial gene expression and wing formation in Drosophila. Curr Biol 1995, 5:1437-1448.
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Diaz-BenjumeaFJ, Cohen SM: Serrate signals through Notch to establish a Wingless-dependent organizer at the dorsal/ventral compartment boundary of the Drosophila wing. Development 1995, 121:4215-4225.
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Speicher SA, Thomas U, Hinz U, Knust E: The Serrate locus of Drosophila and its role In morphogenesis of the wing imaginal discs: control of cell proliferation. Development 1994, 120:535-544.
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Gu Y, Hukriede NA, Fleming RJ: Serrate expression can functionally replace Delta activity during neuroblast segregation in the Drosophila embryo. Development 1995, 121:855-865.
56. Calof AL: Intrinsic and extrinsic factors regulating vertebrate A neurogenesls. Curt Opin Neurobiol 1995, 5:19-27. review that takes a broader perspective than the present one, examining the whole system of controls of vertebrate neurogenesis, including proneural and neurogenic genes. 57.
Lewis J: Rules for the production of sensory cells. Ciba Found Symp 1991, 160:25-39.
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Hartenstein V: Early neurogenesls in Xenopus: the spatiotemporal pattern of proliferation and cell lineages in the embryonic spinal cord. Neuron 1989, 3:399-411.
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KimmelCB, Warga RM, Kane DA: Cell cycles and clonal strings during axis formation of the zebrafish central nervous system. Development 1994, 120:265-276.
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MartinA, Langman J: The development of the spinal cord examined by autoradlography. J Embryo/E.xp Morphol 1965, 14:25-35.
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Couso JP, Martinez Arias A: Notch Is required for wingless signalling in the epidermis of Drosophila. Cell 1994, 79:259-272.
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TakahashiT, Nowakowski RS, Caviness VS: Mode of cell proliferation in the developing mouse neocortex. Proc Natl Acad Sci USA 1994, 91:375-379.
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Greenwald I: Structure/function studies of IIn-12/Notch proteins Curt Opin Genet Dev 1994, 4:556-562.
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Lardelli M, Dahlstrand J, Lendahl U: The novel Notch homologue mouse Notch 3 lacks specific epidermal growth factor repeats and is expressed in proliferating neuroeplthelium. Mech Dev 1994, 46:123-136.
73.
Conlon IRA, Reaume AG, Roasant J: Notch 1 is required for the coordinate segmentation of somites. Development 1995, 121:1533-1545.
74.
Swiatek PJ, Lindsell CE, Del Amo FF, Weinmaster G, Gridley T: Notch 1 is essential for postimplantation development in mice. Genes Dev 1994, 8:707-719.
82. ••
Austin CP, Feldman DE, Ida JA, Cepko CL: Vertebrate retinal ganglion cells are selected from competent progenitors by the action of Notch. Development 1995, 121:3637-3650. Increased numbers of ganglion cells are produced in chick retina when Notch activity is diminished by treatment with antisense oligonucleotides, and decreased numbers are produced when the embryonic retina is infected with a retrovirus carrying a gene for a constitutively active form of Notch. Retinal progenitor cells in vitro are inhibited from differentiating into neurons when co-cultured with Drosophila cells expressing Delta. 63.
64. ••
Dorsky RI, Rapaport DH, Harris WA: Xotch inhibits cell differentiation in the Xenopus retina. Neuron 1995, 14:487-496. Chenn A, McConnell SK: Cleavage orientation and the asymmetric inheritance of Notch1 Immunoreactivity in mammalian neurogenesis. Cell 1995, 82:631-641.
75.
LevitanD, Greenwald h Facilitation of lin-12-medlated signalling
•
by sel-12, a Caenorhabditis elegans S182 Alzheimer's disease
gene. Nature 1995, 377:350-354. A gene identified in C. elegans as a suppressor of a gain-of-function mutation in the Notch homologue/in-12 turns out to be homologous to a human gene implicated in a hereditary form of Alzheirner's disease.
Address Imperial Cancer Research Fund (ICRF) Developmental Biology Unit, Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK e-mail: [email protected] Abbreviations AS-C achaete-scutecomplex bHLH basic helix-loop-helix E(spl)-C Enhancer-of-split complex lIES hairy-and-Enhancer-of-split SMC sensorymother cell Su(H) Suppressor of Hair/ess
Current Opinion in Neurobiology 1996, 6:3-10 © Current Biology Ltd ISSN 0959-4388
Introduction A neuron in a fly or a worm is recognizably the same type of cell as a neuron in a vertebrate; but is it generated in the same way? Has evolution conserved not only the cell type, but also the machinery for its production? At first sight, the answer appears to be no: if we compare the developmental anatomy of the CNS of a vertebrate with that of Drosophila, for example, the structures appear radically different. The vertebrate has a dorsally located neural tube, with neurons differentiating within the neuroepithelium, whereas Drosophila has instead a ventrally located chain of segmental ganglia, with neurons generated from neuroblasts that are derived by delamination from the neuroectoderm. Anatomical appearances can be deceptive, however, and in the past few years it has become increasingly clear that remarkably similar sets of genes are involved in neurogenesis in the different classes of animals. Thus, Drosophila and Caenorhabditis elegans offer a key to the understanding of neurogenesis in vertebrates.
This review focuses on one particular subset of the genes that regulate neurogenesis. The members of this subset are called the neurogenicgenes because, in Drosophila, mutations in them result in massive over-production of
neurons. The first part of this review introduces the neurogenic genes through an outline of their role and mechanisms of action in Drosophila neurogenesis, where there have been some important recent advances. Against this background, the second half of the review will discuss new findings on the role of the neurogenic genes in vertebrate neurogenesis. Neurogenic
genes
and lateral inhibition
in
Drosophila neurogenesis Work over many years in flies and worms [1-3,4",5"] has identified the products of the neurogenic genes as the components of a cell-cell signalling mechanism whereby, in the nervous system, a cell that becomes committed to a neural fate inhibits its neighbours from doing likewise. In this process of lateral inhibition, the receptor for the inhibitory signal is a transmembrane protein encoded by a neurogenic gene called Notch in Drosophila, and the ligand, expressed in the cell delivering inhibition, is another transmembrane protein encoded by a neurogenic gene called Delta in Drosophila. The inhibitory signals are exchanged within groups of cells, so-called proneural clusters, that all have the potential for a neural fate; the role of lateral inhibition is to ensure that only a scattered subset of the cells actually become committed to that fate. The proneural clusters are defined by expression of proneural genes, chiefly the members of the achaete-scute complex (AS-C). The products of the AS-C genes are basic helix-loop-helix (bHLH) transcription factors, which have an initial upstream role as inducers of the expression of neurogenic genes throughout the proneural cluster. T h e y also have a subsequent downstream role, in that their own expression is, in turn, regulated by the lateral inhibition machinery: as cells become committed to a neural fate, their content of AS-C proteins increases, whereas that of their neighbours in the cluster decreases [6]. The heightened expression of AS-C genes in the nascent neural cell is transient, as is the expression of Delta [7,8]. In the embryonic CNS of Drosophila, neuroblasts are generated from the neuroectoderm in successive waves [9], with the proneural and neurogenic genes controlling the proportion of cells that are singled out for this fate in each wave of neurogenesis. Each neuroblast will then undergo repeated asymmetric divisions to generate neurons and/or glial cells (see review by Doe and Skeath, this issue, pp 18-24). In the PNS, the same genes control the singling-out of cells from the epidermis to be sensory mother cells (SMCs; also called sense-organ precursors, or SOPs), each of which will undergo two or more asymmetric divisions to generate the neuronal and supporting cells of a single bristle or other sensillum [2]. In this latter
4
Development
process the neurogenic genes are required a second time, providing lateral inhibition to prevent all the progeny of the precursor from becoming neurons [10]. Outside the nervous system, the same genes govern cell differentiation in tissues as varied as the gut epithelium [11"], the musculature [12], and the Malpighian tubules [13]. In each case, the same lateral inhibition mechanism appears to operate, generating a fine-grained pattern in which individual cells are singled out for commitment to a particular mode of differentiation, while the adjacent cells are inhibited from entering upon that commitment.
Recent work has demonstrated at least one pathway by which lateral inhibition with feedback can come about (Figure 1). Activation of Notch induces expression of genes of the Enhancer-of-split complex (E(spl)-C) [18,19"]; these code for repressive b H L H proteins that act, in conjunction with the co-repressor Groucho [20,21"], to downregulate expression of genes of the AS-C [22°]; the AS-C genes, in turn, code for activator b H L H proteins that promote expression of Delta [23]. The net effect is that Notch activation reduces Delta expression by reducing AS-C expression [22"].
How to make neighbours different
Asymmetric cell division The other mechanism for generating asymmetric cell fates during Drosophila neurogenesis depends on asymmetric cell division: two sister cells adopt different fates because they inherit different quantities of a regulatory protein (or set of proteins) that becomes concentrated at one pole of the parent cell. As reviewed elsewhere ([24"]; see also the review by Doe and Skeath, this issue, pp 18-24), at least two proteins, Numb and Prospero, behave in this way both during the asymmetric divisions of neuroblasts in the CNS and during those of SMCs in the PNS [25-29]. These proteins are necessary and sufficient to confer a neural fate on the daughter that inherits them.
In all the examples mentioned above, and in comparable processes in vertebrate neural development, neighbouring cells have contrasting fates. What makes them behave differently? Why, for example, don't all the cells in a proneural cluster remain locked in a state of mutual inhibition, each cell both delivering inhibition to its neighbours and receiving inhibition from them? Two mechanisms have been proposed, with good evidence in Drosophila for both. They are not mutually exclusive, and both must be borne in mind when considering how neighbouring cells come to adopt different fates during vertebrate neurogenesis. Lateral inhibition with feedback The first mechanism can be called lateral inhibition with feedback. It rests on an elaboration of the basic lateral inhibition mechanism, such that the more inhibition a cell receives, the less it is able to deliver [3,14,15]. In molecular terms, this means that increased activation of Notch in a given cell causes a decrease in the amount or activity of Delta in that cell. In a system of two or more cells, this creates a positive feedback loop (Figure 1), because the cell that delivers more inhibition to its neighbours drives down the level of inhibition that it receives back from them, and thereby still further increases its own ability to deliver inhibition. Thus, any initial a s y m m e t r y - - e v e n a small o n e - - b e t w e e n two adjacent cells will be self-magnifying, leading to a state where one cell predominantly receives inhibition and does not deliver it, while the other predominantly delivers it and does not receive it.
Persuasive evidence for this mechanism has come from studies of the way in which cells are singled out to become SMCs in the developing PNS. For example, at the border of a mutant clone where cells with different numbers of copies of the Delta gene confront one another, it is the cells with more Delta gene copies that become SMCs, whereas their neighbours with less copies are inhibited [15]. Strong evidence for lateral inhibition with feedback has come also from work in C. elegans, where the Notch homologue lin-12 and the Delta homologue lag-2 act in this way to control the genesis of differences between initially equivalent cells in the gonad and elsewhere [3,16",17].
In the case of the SMC, there seems at first to be a paradox, in that Delta-Notch signalling is also required to ensure that the daughters of the SMC become different and do not both become neural [10]. It is not hard to see, however, how lateral inhibition may operate in concert with neural determinants such as Numb and Prospero. Suppose, for example, that in the daughters of the SMC, lateral inhibition operates without feedback, so that the daughters persist in delivering inhibition to one another symmetrically. If the neural determinants are absent, both daughters will remain non-neural; if one daughter inherits the neural determinants, and these make her immune to the inhibitory signal, she alone will become neural; and if Delta-Notch signalling is inoperative, neither daughter will be inhibited, and both will become neural; all this fits the facts. There is indeed evidence suggesting that lateral inhibition between the progeny of the SMC does operate without feedback: the AS-C gene products disappear from the SMC before its first differentiative division and play no part in controlling the fate of its progeny [7], so there cannot be any feedback of Notch on Delta via AS-C genes in this case.
From membrane to nucleus: the mechanism of signal transduction by Notch Regardless of whether lateral inhibition operates with feedback or without, Delta-Notch signalling poses the perennial problem of signal transduction: how does activation of Notch at the cell membrane bring about changes in the pattern of gene expression? Several recent studies, in flies, worms, and vertebrates, together make a strong case for a novel and unusually direct mechanism
Neurogenic genes and vertebrate neurogenesis Lewis
5
Figure 1
Delta-Notch signalling between adjacent proneural cells Cytoplasm ~
.
.
.
.
.
.
.
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DeltaG ~ ~
Cytoplasm IotchlC
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© 1996 Current Opinion in Neurobiology
The Delta-Notch pathway for lateral inhibition according to the current view, depicted for a pair of adjacent proneural cells. Only the components discussed in this review are shown; although the signalling pathway is known to depend on several additional components, their specific roles are still obscure. DNA-binding proteins that function as repressors are marked with a '-', those that function as activators with a '+'. A dashed arrow symbolizes a regulatory link between AS-C genes and Delta that provides for feedback in the lateral inhibition of one cell by the other; Ac/Sc stands for Achaete/Scute proteins - the products of the AS-C. The regulatory links shown probably do not represent the only means by which Notch activation exerts its effects. Some evidence suggests, for example, that the main control of Delta activity during the singling-out of neural cells in Drosophila is post-translational rather than transcriptional [8].
(Figure 1): ligand binding causes cleavage of the Notch protein, releasing from it an intracellular fragment, called NotchlC, which then moves into the nucleus to regulate transcription [30",31",32]. Experiments in mammalian cells show that mutant forms of Notch lacking most of the extracellular domain but retaining the transmembrane domain undergo cleavage to release NotchlC, even in the absence of ligand binding [31"]. In transgenic fly and worm embryos, and in cultured mammalian P19 cells, both NotchlC and these other truncated forms of Notch have a dominant antineurogenic effect, inhibiting neural differentiation in the manner expected if there is constitutive activation of the Notch signalling pathway [32-37]. NotchlC has nuclear localization signals and includes a domain that mediates binding to the product of another neurogenic gene, Suppressor of Hairless (Su(H)) [38",39°]. In Drosophila $2 cultured cells, activation of Notch by Delta leads to a movement of Su(H) protein into the nucleus [38"']. In the nucleus, Su(H) acts by binding to regulatory regions of the E(spl)-C and inducing transcription of E(spl)-C genes [40"], and studies of the homologous genes in mammalian cells indicate that this action of Su(H) depends on the presence of NotchlC bound to it [30"'].
'Activated Notch' constructs--that is, NotchlC and other forms of Notch lacking the extracellular d o m a i n - - a r e powerful tools for the analysis of Notch-dependent signalling mechanisms. For example, they have been used in combination with loss-of-function mutations of other neurogenic genes to discover the epistatic ordering of these genes; these tests confirm that the E(spl)-C lies downstream from Notch, that Delta is upstream from Notch, and that the neurogenic genes mastermind (mare), big brain (bib), neuralized (neu) and almondex (amx) also lie upstream from Notch [ 3 2 ] - - t h a t is, the presence of activated Notch causes an antineurogenic phenotype regardless of whether these genes are functional. T h e precise roles of mam, bib, neu and amx remain a mystery, however.
Neurogenic genes in vertebrates In vertebrates, there have been only two reports of neurogenic genes discovered by the methods of classical genetics: one is called white tail and was found in the Ttibingen zebrafish mutagenesis screen [41"], and the other is called mind bomb and was found in the Boston zebrafish screen [42°]. It is possible that they both are alleles of the same gene. By comparison, the number of vertebrate genes identified by homology with Drosophila
6
Development
neurogenic genes is large. First to be discovered was
Xotch, a Xenopus Notch homologue, now renamed X-Notch-1 [43]. Since then, multiple Notch homologues have been found in rat, mouse, human, zebrafish and chick (reviewed in [5°]). More recently, homologues of Delta have been found [44",45",46",47], as well as homologues of the Delta-related gene Serrate [48"',49"]. There is a whole family of homologues of E(spl)-C b H L H genes [50"',51], known as HES g e n e s - - f o r hairy-and-Enhancer-of-split h o m o l o g u e s - - b e c a u s e they also resemble the Drosophila gene hairy. There is also a family of vertebrate groucho homologues, called TLE genes [52,53]. Su(H) turns out to have as its homologue a vertebrate gene previously identified in another context as coding for a transcription factor variously called RBP-J~ CBF1 or KBF2 [39",54,55]. T h e vertebrate achaete-scute homologues (ASH genes) are again numerous; their role in vertebrate neurogenesis is reviewed elsewhere [56"], and is a large topic that will not be covered here except in passing.
Vertebrate Notch and Delta homologues and lateral inhibition in primary neurogenesis In vertebrates, neurons are generated in at least three distinct ways: within the central neuroepithelium that forms the neural plate and neural tube; by migration from the neural crest; and by delamination from the cranial placodes. Although placodal neurogenesis shows the most intriguingly close anatomical parallels with insect neurogenesis [57], I shall concentrate here on central neurogenesis, on which most of the recent relevant research has been done. In the central neuroepithelium, the first neurons appear as scattered postmitotic cells in a number of distinct regions (comparable with proneural clusters in the insect neuroectoderm), where they are mingled with undifferentiated proliferative neuroepithelial cells. T h e pattern is most strikingly displayed in the neural plate of amphibians and fish, where production of a set of primary neurons is separated by a time-lag from production of subsequent sets of neurons [58,59]. Notch-l, and possibly other Notch homologues, are expressed by all the cells in the regions where primary neurogenesis is occurring. Meanwhile, expression of the Delta homologue Delta-1 is r e s t r i c t e d - in Xenopus and chick, at l e a s t - - t o a scattered subset of cells that have ceased dividing and appear to be the nascent neurons [45",46"]. This suggests that Delta-Notch signalling is operating as in the fly to single out cells for a neuronal fate. Gene functions can be tested directly in Xenopus by injecting RNA into the embryo at the two-cell stage. When RNA coding for full-length Deha-1 protein is injected, the production of neurons is inhibited [45°']: forced expression in all cells of a gene normally expressed only in nascent neurons inhibits all cells from becoming nascent neurons. This behaviour, at first sight paradoxical, is the hallmark of a gene whose function is to deliver
lateral inhibition: normally, isolated cells express the gene and inhibit their neighbours, from whom they receive no inhibition; but when all cells are forced to express the gene, they all inhibit one another. Injection of RNA coding for a modified Delta-1 protein with a truncated intracellular domain has a contrary effect, causing excess production of neurons, as though it is blocking the normal delivery of inhibition by the endogenous Deha-1. Injection of RNA coding for activated N o t c h - - e i t h e r NotchlC, or Notch-1 with a truncated extracellular d o m a i n - - n o t only prevents the production of neurons, as expected if NotchlC inhibits commitment to a neural fate, but also inhibits expression of the endogenous Delta-1 gene [45"°]. This implies the existence of a feedback in the lateral inhibition system, of the type described above for Drosophila (see Figure 1): a cell expressingDelta-1 more strongly than its neighbours will not only tend to inhibit them from becoming neurons, but will also inhibit them from delivering inhibition reciprocally and so will reinforce its own advantage. In this way, starting perhaps with uniform low-level expression of Delta-1 or with uniform expression of some other Delta homologue, cells within an initially homogeneous population may become singled out for a neural fate.
Vertebrate Notch and Delta homologues in later neurogenesis Is this conserved machinery of lateral inhibition with feedback a peculiarity of primary neurogenesis, or does it operate in subsequent neurogenesis too? After the first neurons in a vertebrate have been generated, the undifferentiated neuroepithelial cells of the embryonic CNS serve as stem cells for the production of further neurons. Over a period of many days, they continue to divide, with a proportion of their progeny remaining proliferative and a proportion withdrawing from the cell cycle and embarking on differentiation as neurons [60,61]. A layered structure soon develops, with the cell bodies of the proliferative cells confined to the ventricular zone (at least in hindbrain and spinal cord), and the postmitotic nascent neurons moving out of this region into the mantle zone as they begin to differentiate. Studies in the chick show that Notch-1 meanwhile is expressed in apparently all cells in the ventricular zone, while Delta-1 is expressed in a non-dividing subset of cells that are scattered within the ventricular zone and appear, once again, to be nascent neurons [49"]. Expression of both genes becomes switched off by the time the nascent neuron moves from the ventricular zone to the mantle zone. All this amounts to strong circumstantial evidence for continuing control of neurogenesis by the same Delta-Notch signalling mechanism as in Drosophila. More direct proof comes from experiments on the developing chick retina [62"']. Activated Notch, carried into the cells by a retroviral vector, inhibits production of neurons; conversely, antisense oligonucleotides targeted against Notch-1 cause
Neurogenic genes and vertebrate neurogenesis Lewis
increased production of neurons. And when the retinal cells are co-cultured with Drosophila cells expressing Delta, they are inhibited from giving rise to neurons. Similarly, in the Xenopus retina, activated Notch inhibits neuron production [63]. T h e neuroepithelium of the developing vertebrate CNS can thus be compared with the neuroectoderm of Drosophila, and the nascent neurons of the one with the neuroblasts of the other. In both cases, scattered cells within a neuroepithelium become singled out for a neural fate, transiently express high levels of Delta, and segregate from their close neighbours expressing Notch, which are inhibited from differentiating and remain to generate subsequent batches of neural cells. There are, however, some important differences: in the vertebrate CNS, the nascent neurons have stopped dividing, whereas in Drosophila, the neuroblasts continue to divide; and in the vertebrate CNS, the nascent neurons stay within the neuroepithelium, whereas in Drosophila, the neuroblasts escape from it. Vertebrate placodal neurogenesis is more similar to insect neurogenesis in these respects, and indeed Notch and Delta homologues are expressed in placodal epithelium at sites of neurogenesis (J Adam, D Henrique, A Myat, unpublished data). It is not clear what part, if any, the Delta-Notch signalling mechanism plays in creating the differences among neuronal and glial cell types in the vertebrate CNS, and there is reason to suspect that asymmetric cell divisions, as well as lateral inhibition with feedback, are important. In the developing cerebral cortex of a mammal, cells in the proliferative zone are seen to divide symmetrically when generating pairs of daughters that both remain in the proliferative zone, but asymmetrically and with a differently oriented mitotic spindle when one of the daughters is destined to emigrate from the proliferative zone (presumably to become a neuron) [64"']. In the latter case, the asymmetry is marked chemically by the distribution of Notch-1 protein; puzzlingly, this becomes concentrated at the basal pole of the dividing cell and is segregated to the emigrating (neuronal) daughter (as one might expect for N u m b and Prospero proteins, rather than Notch, in corresponding circumstances in an insect).
Alternative ligands for Notch From the examples studied so far, it seems that DeltaNotch signalling has a universal role in the control of vertebrate neurogenesis. There is, however, at least one important qualification: Drosophila possesses another gene, Serrate, that also codes for a Notch ligand, and it seems that homologues of Serrate also have a role as Notch ligands during vertebrate neurogenesis in certain regions of the nervous system. Serrate is structurally related to Delta and contains a similar highly conserved Notch-binding domain (the DSL domain [65]), but it acts in different circumstances--for example, in Drosophila, it does not control neurogenesis, but controls the creation of the wing
7
margin, a specialized band of tissue with a key organizing role in the growth of the wing imaginal disc [66-68]. In artificial conditions, Serrate can nevertheless serve to regulate neurogenesis [69]. In the rat, a homologue of Serrate, called Jagged, is expressed in the CNS in restricted domains of the ventricular zone, and, in an in vitro assay, is capable of activating Notch-1 to control gene expression [48"']. Studies of the corresponding chick gene, called C-Serrate-I, show that it is expressed in the ventricular zone of the neural tube in narrow stripes that extend from the anterior part of the hindbrain all the way down the spinal cord [49"]. These stripes correspond exactly to gaps in the expression domain of C-Delta-l, and the cellular pattern of C-Serrate-1 expression within them suggests that C-Serrate-1 may here act instead of C-Delta-1 as a Notch ligand. T h e analogy with the fly, however, would suggest that C-Serrate-1 is more than just a stand-in for C-Delta-l, and that something special is happening at these sites. A still richer vein of speculation is opened up if, as one school of thought has argued, Notch proteins act as receptors not only for members of the Delta/Serrate family, but also as receptors for the signalling protein Wingless and its vertebrate relatives, the Wnts [66,70].
Vertebrate E(spl) homologues: lessons from transgenic mice Notch and Delta are not the only neurogenic genes for which there is direct evidence of a conserved function in neurogenesis. T h e mouse hairy-E(spl) homologue HES-1, for example, is also expressed throughout the ventricular zone, and its role there has been tested both by overexpressing the gene using a retroviral vector and by knocking it out by homologous recombination [50",51]. In accordance with the function of E(spl)-C genes in Drosophila, cells forced to express HES-1 persistently are inhibited from adopting a neuronal or glial fate and remain in the ventricular population, eventually becoming ependymal cells or dying. Conversely, in the HES-I knockout animal, some classes of neurons are generated prematurely. These effects, again as expected by analogy with the fly, correlate with changes in the expression of AS-C homologues. Knockouts of Notch-I and RBP-JK/Su(H) have been less informative, perhaps because of genetic redundancy [71,72]: in both cases, the mutant embryos die half way through gestation, and the relatively mild abnormalities seen in the cytoarchitecture of the neural tube at this stage are difficult to decipher [55,73,74].
Conclusions Lateral inhibition mediated by Delta-Notch signalling appears as a key mechanism in the control of neurogenesis. It seems to be a general principle that neurons are generated, and subsequently function, within a matrix of cells of other types; lateral inhibition provides the means to single them out from their neighbours for their special fate. T h e recent discoveries have revealed in outline how the signalling pathway works, and have shown, for several
8
Development
of the main components, that it is similar in vertebrates, flies and worms. T h e course of evolution has indeed conserved not only the neuron as a specialized cell type, but also a major part of the developmental mechanism that gives rise to it. T h e task now is to pursue the many components of the Delta-Notch signalling pathway that remain mysterious (e.g. see [75"]), to find out more about how this mechanism of lateral inhibition is combined with other developmental controls, and to define precisely the cell-fate decisions that it governs, not only in the nervous system but also outside it.
Acknowledgements I thank D lsh-Horowicz for comments on the manuscript, the members of his lab and my lab for many discussions, and P Simpson, I: Guillemot, S Bray, R Kopan, F Schweisguth, Y-J Jiang, A Schier, C Cepko, 1 Greenwald, W Driever and A Martinez-Arias for copies of their papers in advance of publication. I am indebted to the Imperial Cancer Research Fund for support.
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