seminars in CELL & DEVELOPMENTAL BIOLOGY, Vol 9, 1998: pp. 583]589 Article No. sr980266

Notch signalling and the control of cell fate choicesin vertebrates

Julian Lewis

Signals delivered via Notch and its ligands Delta andSerrate control developmental choices made by individualcells according to the states of their immediate neighbours.Lateral inhibition mediated by Notch governs neurogenesis.In the inner ear, it generates fine-grained patterns ofcontrasting cell types. In stem-cell systems, it may regulatethe decision to differentiate. Notch signalling can createspecialised cells at gene expression boundaries, as at thelimb-bud apex. It is crucial for segmentation of the mesoderminto somites, for development of skin appendages, and formany other functions that we do not yet understand.

Key words: Delta r lateral inhibition r neurogenesis rNotch r vertebrates

Q1998 Academic Press

Introduction

VERTEBRATES HAVE MULTIPLE HOMOLOGS of Notch andof its ligands Delta and Serrate, and these are ex-pressed, in varying combinations, at many sites in thedeveloping vertebrate body. When components ofthe Notch signalling pathway are genetically da-maged or perturbed, many things go wrong. Thechallenge is to identify not just where, but how andwhy the effects occur. Precisely what cell-fate deci-sions does the Notch signalling machinery govern?How does it govern them? What part does it play indefining the spatial pattern of cell differentiation? Ina few cases, these questions have clear answers; inothers, we have only hints.

From the Vertebrate Development Laboratory, Imperial CancerResearch Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK

Q1998 Academic Press1084-9521r98r060583q07 $30.00r0

Notch signalling and the control of fine-grained pattern: some principles

Before discussing specific examples, it is helpful toconsider some principles that one may expect toapply to Notch signalling generally.1

v First, both Notch and its ligands Delta andSerrate are integral membrane proteins andgenerally transmit signals only between cells indirect contact. In many cases, the interactingneighbours are cells with a similar developmen-tal history and similar developmental potential}that is, they are members of the same equiva-lence group.

v Second, activation of Notch has a direct andimmediate effect on gene expression, mediatedby the detached intracellular domain of Notchitself, acting as a transcriptional regulator in thenucleus.2,3 Thus Notch signalling can readilythrow genetic switches that determine choicesof cell fate.

v Third, activation of Notch in a given cell fre-quently regulates production of Notch ligandsby that cell. Because the level of Notch activa-tion in the cell depends on the level of ligandexpression in its neighbours, and vice-versa, thisgives rise to feedback loops that correlate thefates of adjacent cells and control the fine de-tail of the spatial pattern of differentiation.4,5

In the most famous case, that of lateral inhibition,activation of Notch inhibits production of the Notchligand. Consequently, a cell that produces more lig-and forces its neighbours to produce less; and thisenables the cell to increase its ligand productioneven further, because it receives a weakened inhibi-tory signal back from its neighbours. The effect ofthis feedback loop is to drive neighbouring cells intodifferent developmental pathways: any initial differ-ence between them, whether it be small and stochas-tic or large and predictably imposed, is intensifiedand maintained.

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Notch signalling can, however, also be responsiblefor the contrary phenomenon, lateral induction, asrecent work on the Drosophila wing margin has shown:here, activation of Notch promotes production ofNotch ligands.6,7 Thus a cell expressing raised levelsof ligand stimulates its neighbours to do likewise:instead of shouting one another down, the cells eggone another on. Whereas lateral inhibition tends todrive a group of initially equivalent cells to developinto a pepper-and-salt mosaic of cells in differentstates, lateral induction can be expected to do exactlythe opposite: it will cause cells within such a group tomake their cell-fate choices cooperatively, preventingthe occurrence of pepper-and-salt mixtures andfavouring all-or-none behaviour and the formation ofsharply defined boundaries of gene expression.

It should be emphasized that the distinctionbetween lateral inhibition and lateral induction, asdefined here, does not lie in whether activation ofNotch inhibits or induces cell differentiation, but inwhether it inhibits or induces expression of theNotch-activating ligand. If ligand production is inhib-ited, neighbouring cells will be driven to differ fromone another; if ligand production is induced, theywill be driven to be similar. This is true regardless ofwhether Notch activation promotes or inhibits celldifferentiation. The hallmark of lateral inhibition,therefore, is fine-grained variegation in the patternof expression of a Notch ligand; and the hallmark oflateral induction is local uniformity and cooperativityin its expression.

Lateral inhibition in the control of vertebrateneurogenesis

In vertebrates, as in Drosophila, the most thoroughlyanalysed example of Notch signalling is in the con-trol of neurogenesis. Notch signalling here operatesbetween cells within an equivalence group, mediateslateral inhibition, and controls the commitment todifferentiate.

In the neural tube}the rudiment of the vertebratecentral nervous system}neurons are generated fromdividing precursors whose cell bodies lie in a prolifer-ative zone close to the lumen of the tube. When aprogenitor divides, its progeny have a choice. Eachdaughter can either remain a progenitor or become

committed to differentiate as a neuron; in the lattercase, it withdraws from the cell division cycle andmigrates out into the mantle zone of the neuroep-ithelium, where it differentiates. Notch1 is expressedthroughout the proliferative zone; Delta1 is expressedin the outer part of that zone, in a scattered subset ofcells.8 From their location and their non-dividingcharacter, these Delta1-expressing cells can be identi-fied as nascent neurons.9 Analogy with Drosophilaneurogenesis immediately suggests what is happen-ing: the nascent neurons, by expressing Delta1, de-liver lateral inhibition to the Notch1-expressing pro-genitors in contact with them, so as to prevent theseprogenitors from differentiating prematurely intoneurons and from expressing Delta1. This interpreta-tion of the gene expression patterns has been amplyconfirmed by experiments in Xenopus,10 ] 12 chick,13,14

mouse15 and zebrafish,16 ] 18 which demonstrate allthe features of the lateral inhibition mechanism de-scribed above, including inhibitory regulation of Deltagenes by Notch activation. I will outline here just oneof these experimental studies, because it highlights akey role of Notch signalling in regulating the tem-poral as well as the spatial pattern of development.

To generate the vast numbers of neurons that thevertebrate central nervous system requires, neuroge-nesis has to be prolonged for many days, weeks, oreven months, during which the stem-cell-like progen-itors in the wall of the neural tube have to continuethrowing off differentiated progeny. The embryonicchick retina}an outgrowth of the neural tube}is aconvenient model system in which to study theprocess. A retroviral vector can be used to misexpressDelta1 or a dominant-negative derivative of Delta1,called Delta1dn, and thereby to activate or block theNotch signalling pathway.13,14 Normally, only thenascent neurons, scattered among the dividing pro-genitors, express Delta1. Where all cells are forced toexpress Delta1, neurogenesis is suppressed and all thecells remain as progenitors. Conversely, where all thecells are forced to express Delta1dn, all the cellsdifferentiate prematurely as neurons and no dividingprogenitors remain. Thus lateral inhibition mediatedby Delta]Notch signalling is the mechanism that reg-ulates the choice between remaining as a progenitorand embarking on differentiation. The inhibitiondelivered from the differentiating progeny to theirdividing progenitors acts homeostatically to limit theproportion of cells that differentiate, thereby main-taining a balanced mixture of the two classes of cellsso that neurogenesis can continue.13

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This finding in the retina raises an important gen-eral question about the role of Notch signalling in

Ž .stem-cell systems see below .

Lateral inhibition as a spatial patterningmechanism: the inner ear

In Drosophila, the development of sensory bristlesprovides another well-known paradigm of lateral in-hibition mediated by Notch.19 ] 21 These mechanosen-sory organs on the surface of the insect have acounterpart in the mechanosensory patches in theinner ear of a vertebrate, and there are many devel-opmental parallels between the two structures.22 Inparticular, Notch signalling seems to operate repeat-edly to generate the pattern of different cell types inthe ear’s sensory patches, just as it does in the forma-tion of the bristle. Thus in the ear of the chickembryo, expression of Delta1 in scattered cells, againsta background of uniform Notch1 expression, fore-shadows the emergence of neuroblasts from the earepithelium; and a little later, expression of Delta1 innascent sensory hair cells foreshadows formation ofthe alternating mosaic pattern of sensory hair cellsand supporting cells found in a mature sensory patch.As in the neural tube, the gene expression patternssuggest that lateral inhibition is at work}in particu-lar, that the nascent hair cells, by expressing Delta1,inhibit their neighbours from becoming hair cellsand force them to be supporting cells instead.

The mind bomb mutant zebrafish corroborates thishypothesis. Mind bomb has a neurogenic phenotype:in the central nervous system, it produces primaryneurons in excess, in exactly the way that would beanticipated if there were a failure of Delta]Notchsignalling.23,24 This is accompanied by overexpressionof Delta genes at the sites of neurogenesis. The over-production of neurons despite overexpression of Deltagenes implies that the cells in the mutant neuralplate have lost their susceptibility to lateral inhibi-tion: they are insensitive to Delta]Notch signalling.When the ear of the mind bomb mutant is examined,it shows exactly the defect that would be predicted:neurons are overproduced, and, most dramatically,the sensory patches consist of nothing but hair cells,which are generated prematurely and in vast excessat the expense of supporting cells.25 Lateral inhibi-tion mediated by Delta]Notch signalling thereforeappears to be the mechanism that organises the nor-mal hair-cellrsupporting-cell mosaic.

In the ear, as is common elsewhere, expression ofNotch and Delta family members goes hand in handwith expression of Serrate and fringe family members,in closely related but non-identical patterns. Thus thenascent hair cells switch off expression of Delta genesas they differentiate, but maintain expression of a

Ž 25Serrate homolog serrateB in the zebrafish, Jagged226 .in the mouse ; this alternative Notch ligand may

serve to keep the supporting cells in their inhibitedstate. Another Serrate homolog is also expressed inthe developing sensory patches, in yet another pat-tern, marking out the location of the patches by itsuniform expression within them throughout theirearly development.22 The individual functions of allthese related components are not easy to disentangle.

Similar puzzles are encountered in many othervertebrate tissues, where one likewise finds homologsof Notch, Delta and Serrate}often several homologsof each}expressed together.8,27,28 The endothelialcells that line blood vessels, for example, expressNotch1, Notch4, Delta1, Serrate1, and possibly othermembers of these families,8,29 and there is evidencefrom knock-outs and antisense experiments that thegenes here have more than a decorative role.30,31 Butprecisely what aspect of endothelial cell behaviour isgoverned by Notch signalling? This is still a mystery.

Lateral inhibition and the control of stem cellfunction: skeletal muscle growth andhaemopoiesis

By definition, stem cells divide to give a mixture ofprogeny, some of which remain as stem cells whileothers embark on differentiation. What drives adja-cent progeny in such a system to follow differentdevelopmental pathways, so that stem cells and dif-ferentiating cells both continue to be produced inproperly controlled proportions? In the central ner-vous system, as explained above, Notch signalling hasthis role: each nascent differentiating cell deliverslateral inhibition, preventing its neighbours fromdifferentiating in the same way at the same time.Does the same mechanism operate elsewhere, in anyof the many other vertebrate tissues that depend onstem cells for growth and renewal? As yet there areno firm answers. In the epidermis, for example, ho-mologs of Notch and its ligands are expressed,27,32,33

Ž .but their role if any in the control of stem cellbehaviour is unresolved. Muscle and bone marrow,however, offer some hints.

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Developing skeletal muscle is a mixture of differ-entiated muscle cells and dividing myoblasts, whichbehave as stem cells for muscle growth and regenera-tion. Scattered cells in this tissue express Delta1andror Serrate2, and it is possible that these aremyoblasts that are becoming committed to differen-tiate.34 In myoblastic cell lines, activation of Notchinhibits differentiation.35 ] 37 Skeletal muscle, there-fore, is a candidate for regulation of stem-cellcommitment by lateral inhibition as in the centralnervous system.

Haemopoietic stem cells present a different pic-ture. They express Notch1 and Notch2 and sit incontact with stromal cells that express the Serratehomolog Jagged1. Exposure of haemopoietic stemcells to Jagged1 in culture causes an increased pro-portion of them to retain a stem-cell- likecharacter.38 ] 40 The suggestion in this case, therefore,is that inhibition via Notch regulates stem-cellcommitment, but that the inhibition comes fromstromal cells, not from differentiating members ofthe haemopoietic population itself. When a daughterof a haemopoietic stem cell leaves the stromal nest, itloses the Notch-mediated signal that keeps it in astem-cell state.

Notch signalling and the exploitation of geneexpression boundaries: the limb-bud apex

Where two non-equivalent groups of cells confrontone another, Notch signalling from one group to theother can confer a distinctive character on a band ofcells along the boundary between the two popula-tions. In Drosophila, this happens in the wing imagi-nal disc at the wing margin, where ventral cells ex-pressing Notch and Delta confront dorsal cells ex-pressing Notch, Serrate and fringe. The result is theformation of specialised margin cells that producesignals organizing wing outgrowth. In vertebrates, thesame thing happens in a remarkably similar way atthe apex of the limb buds, where ventral ectodermalcells expressing Notch1 and probably Delta1 confrontdorsal ectodermal cells expressing Serrate2 and Radi-cal fringe. The result is formation of the specialised

Ž .cells of the apical ectodermal ridge AER , whichproduce signals organising outgrowth of the verte-brate limb bud.41,42 This phenomenon, well reviewedelsewhere,43 is perhaps the most intricate of theexamples where Notch signalling controls vertebratepattern formation in a way we can claim to under-stand.

Notch signalling and the creation of geneexpression boundaries: somite formation

It is one thing to exploit an existing boundary tomake something special happen there, another thingto create a sharp boundary where none existed be-fore. Somite formation is a prime example ofboundary creation in the latter sense, and it is certainthat Notch signalling has a key role in the process. Itis not so clear, however, precisely what that role is.

Somites form sequentially, from head to tail, bysegmentation of the presomitic mesoderm.44,45 Deltafamily members, along with Notch1, are strongly ex-pressed in the presomitic mesoderm}posteriorly, inthe most immature region, in broad domains withblurred borders; more anteriorly, in more crisplydefined, narrower stripes such that the cleft betweenone somite and the next appears to form at the sharpgene expression boundary where cells that express aDelta gene confront cells that do not.17,46,47

Underlying the whole process of segmentationthere is a temporal oscillation, manifest in the cyclicexpression of the hairy1 gene in the presomitic me-soderm, and coupled to the Delta]Notch signallingpathway through oscillations in expression of Lunaticfringe.48 ] 51 When the Delta]Notch signalling pathwayis blocked by mutations or otherwise interfered with,the clefts between somites form irregularly or not atall.16,23,31,47,52,53 The segmentation defect goes withabnormalities in the Delta expression pattern: thenormal sharp expression boundaries fail to develop,and according to at least some accounts54 the level ofexpression in the presomitic mesoderm is generallyreduced. It seems, therefore, that sharp boundariesof Delta expression, or of Notch activation, are neces-sary to guide somite segmentation, and that theseboundaries themselves are made sharp by a mecha-nism that depends on Delta]Notch signalling. Thismay be a case where Notch mediates, not lateralinhibition, but lateral induction, coordinating levelsof Notch activation in neighbouring cells and en-abling them to create a sharp gene expression

Ž .boundary a pattern discontinuity where no suchdiscontinuity existed before.

Notch signalling in cooperative cell clusters:the formation of skin appendages

Somitic mesoderm is not the only tissue where verte-brates switch on Delta gene expression strongly anduniformly in a group of adjacent cells, as though

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coordinated by lateral induction. Similar expressionpatterns are seen, for example, in the skin, wheregroups of dermal cells express Delta1 as they aggre-gate to form hair or feather papillae. The localisationof Delta1 expression is necessary for the developmentof the skin appendage:32,33 where all cells in thedermis are artificially forced to express Delta1, noappendages form. But it remains to be discoveredwhy this should be so, and what function is per-formed by the changing patterns of expression ofNotch, Delta and Serrate family members in the dermisand epidermis.

Conclusion

This review has emphasised the central role of Notchsignalling in controlling the commitment to differ-entiate and the choice of differentiated cell fate invertebrate tissues. The examples discussed so far areonly a small sample of the cases where, to judge fromgene expression patterns and analogies with inverte-brates, we can expect Notch signalling to play a part.Other vertebrate cells for which there is evidence ofcontrol by Notch signalling include such diverse types

Ž 55.as T cells well-reviewed elsewhere , oligodendro-cytes,56 fat cells,57 mammary gland epithelium58 andvascular smooth muscle.59 Moreover, it is likely thatNotch also acts in other ways, not only as a regulatorof gene expression. In Drosophila, Notch protein ispresent, for example, on the surfaces of mature neu-rons, where it is involved in growth-cone guidance.60

Notch and its ligands on other cell surfaces may actas adhesion molecules to control formation and dis-solution of epithelial organisation.61

The role of Notch signalling in vertebrates is alarge topic, and most of it remains to be explored.

Acknowledgements

I thank my colleagues for many discussions, Alastair Mor-rison and David Ish-Horowicz for comments, and the Im-perial Cancer Research Fund for funding.

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