REVIEW

Mechanisms of Notch signaling: a simple logic deployedin time and spaceDomingos Henrique1,* and François Schweisguth2,3,*

ABSTRACTMost cells in our body communicate during development andthroughout life via Notch receptors and their ligands. Notchreceptors relay information from the cell surface to the genome viaa very simple mechanism, yet Notch plays multiple roles indevelopment and disease. Recent studies suggest that thisversatility in Notch function may not necessarily arise from complexand context-dependent integration of Notch signaling with otherdevelopmental signals, but instead arises, in part, from signalingdynamics. Here, we review recent findings on the core Notchsignaling mechanism and discuss how spatial-temporal dynamicscontribute to Notch signaling output.

KEY WORDS: Cell fate, Notch, Patterning, Signaling, Stem cell

IntroductionNotch is a cell surface receptor that provides Metazoans with anexquisitely simple cell-cell communication device (Artavanis-Tsakonas et al., 1999; Bray, 1998; Kopan and Ilagan, 2009). Itsectodomain can read information about the state of neighboringcells, as it recognizes ligands that are expressed at their surface,whereas its intracellular domain acts, upon activation, as atranscription regulator that adjusts the cell state according to thestate of neighboring cells. Signaling from the cell surface to thegenome is direct, linear and devoid of signal amplification (Fig. 1).It is also transient, as each receptor is used only once and thereleased intracellular domain is short-lived. Despite this inherentsimplicity, the study of Notch signaling is often viewed asdiscouragingly complex and it is not uncommon to hear scientistshaving to reluctantly consider the role of Notch in their favoriteexperimental system. Moreover, Notch signaling is known tofunction throughout development in a plethora of cells, tissues andorgans, regulating many cellular behaviors in a context-dependentmanner, ranging from differentiation, proliferation, death tomigration. This pleiotropy can often mask the operation of asimple signaling mechanism. In addition, and perhaps notsurprisingly, many factors have been shown to interact in acontext-specific manner with Notch. Although this may suggest theexistence of a very large number of context-specific regulators ofNotch signaling, blurring the simplicity of this signaling device, asimpler interpretation may be that Notch signaling dynamics arehighly sensitive to perturbations in a wide range of cellular

processes. In this Review, we discuss recent findings that supportthis perspective and are likely to be of general relevance. ThisReview will not cover the many specific roles that Notch signalingplays in developmental processes (Artavanis-Tsakonas et al., 1999;Bray, 1998). Likewise, we will not discuss the consequences ofNotch alterations in human disease, but refer the reader to otherreviews on these topics (Mašek and Andersson, 2017; Siebel andLendahl, 2017).

Receptor activation by mechanical allosteryThe core mechanism of Notch receptor activation is now relativelywell understood (Bray, 2006; Gordon et al., 2008; Kopan andIlagan, 2009) (Fig. 2A). Receptors in a given cell are activated in ajuxtacrine manner by cell surface ligands from neighboring cells ina process known as ‘trans-activation’ (‘trans’ refers here to the factthat signaling ligands and activated receptors are present in distinctcells). In the absence of ligand binding, receptors are in an inactivestate. However, receptor trans-activation following ligand bindingleads to a structural change in the Notch receptor that renders anotherwise buried cleavage site (S2) accessible to metalloproteasesof the ADAM/TACE family (Gordon et al., 2007, 2015; Tiyanontet al., 2011; Weng et al., 2004). Cleavage at this S2 site thengenerates a membrane-tethered form of Notch that is further cleavedby the γ-secretase complex (Mumm et al., 2000; Struhl and Adachi,2000) to release the Notch intracellular domain (NICD). NICDlocalizes to the nucleus, in which it associates with the sequence-specific DNA-binding protein CSL to regulate gene expression(Lecourtois and Schweisguth, 1998; Schroeter et al., 1998; Struhland Adachi, 1998) [note that CSL is an acronym for the names ofthis conserved protein in humans, flies and worms: CBF1 (alsoknown as RBPJ), Su(H) and LAG-1]. Thus, the stepwise proteolyticcleavage of Notch provides a simple, direct and irreversiblemechanism to relay an extracellular signal to the genome.

A key step in pathway activation is the ligand-induced structuralchange in the Notch receptor to make the S2 site accessible.However, although ligand binding is necessary for Notch trans-activation, it is not sufficient, and it is thought that a mechanicalforce is crucial to unmask the S2 site (Fig. 2A). This idea of apulling force exerted onto Notch receptors by signal-sending cellscan be traced back to experiments using Drosophila S2 cells,designed to test whether Notch interacts at the cell surface with itsligand Delta (Fehon et al., 1990). These cell-cell adhesion assaysnot only showed that Notch and Delta mediate heterophilicadhesion, but further revealed that the receptor is transferred fromNotch-expressing cells into Delta-expressing cells. This transfer isdependent on Delta endocytosis, indicating that Notch is trans-endocytosed. This led to the proposal that ligand endocytosis drivesreceptor dissociation and activation via mechanical pulling (Parkset al., 2000). Soon afterwards, structural, modeling and humangenetics studies revealed that the S2 cleavage site is buried withinthe negative regulatory region (NRR) of Notch, and that receptors

1Instituto de Histologia e Biologia do Desenvolvimento and Instituto de MedicinaMolecular, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egaz Moniz,1649-028 Lisboa, Portugal. 2Institut Pasteur, Department of Developmental andStem Cell Biology, F-75015 Paris, France. 3CNRS, UMR3738, F-75015 Paris,France.

*Authors for correspondence (henrique@medicina.ulisboa.pt;francois.schweisguth@pasteur.fr)

D.H., 0000-0001-8869-1894; F.S., 0000-0001-8888-9390

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with mutations that are predicted to destabilize the NRR, henceexposing the S2 site at the protein surface, are hyperactive in aligand-independent manner (Gordon et al., 2007, 2015; Tiyanontet al., 2011; Weng et al., 2004). In addition, biophysicalexperiments demonstrated that applying pulling forces in therange of those produced by endocytosis (∼5-10pN) is sufficientfor receptor activation, and that ligand-mediated allostery andoligomerization are not essential for force-dependent shedding(Gordon et al., 2015; Luca et al., 2017; Meloty-Kapella et al., 2012;Seo et al., 2016; Wang and Ha, 2013). More recently, the lifetime ofNotch-ligand complexes was found to increase as a function ofapplied force, reaching a maximum at ∼10 pN for jagged canonicalNotch ligand 1 (Jag1). This ‘catch-bond’ property, wherebydissociation lifetime increases when force is applied to the bond,suggests that complex stability increases as the ligand becomesendocytosed (Luca et al., 2017). At the structural level, the pullingforce may first induce a rotation within the Notch-binding region ofthe ligand, which adopts an elongated conformation that increases theligand-receptor contact interface. The resulting increased bindingmay then stabilize the complex under tension and transmit force to theNRR (Luca et al., 2017). Together, these data provide strong evidencethat Notch activation is a mechanosensitive process that requires apulling force to expose an otherwise buried S2 cleavage site, and thatthis force might arise from the endocytosis of bound ligands(Lovendahl et al., 2018) (Fig. 2A).Testing the mechanical pulling model in vivo has remained

challenging. However, an elegant genetic mosaic strategy wasrecently designed to probe force generation by ligand-dependentendocytosis using chimeric ligand-receptor binding pairs inDrosophila (Langridge and Struhl, 2017). In this assay, signalingbetween heterologous ligand-receptor pairs was monitored alongartificial clone borders. One such chimeric ligand-receptor pairinvolved the follicle-stimulating hormone (FSH; fused to Delta) andthe extracellular domain of its receptor (FSHR, fused to Notch) andwith the NRR of Notch replaced with a force sensor domain derivedfrom von Willibrand factor (vWF) (Fig. 2B). The unfolding of thisdomain, which is termed the A2 domain, requires a force greaterthan 8 pN. When tested in vivo, the chimeric FSHR-A2-Notchreceptor was not activated by Delta-FSH. This suggests that the A2

domain does not allow for ligand-dependent cleavage of thereceptor, presumably because the force produced by the endocytosisof Delta-FSH is below 8pN. In contrast, chimeric receptors withmutant versions of the A2 domain that display lower thresholds forunfolding appeared to be processed in a ligand-dependent manner(Fig. 2B). This observation strongly suggests that the endocytosis ofDelta-FSH provides a pulling force sufficient to unfold these mutantversions of the A2 domain. Although activation of the FSHR-A2-Notch receptor must involve cleavage, it is not clear, in the absenceof the NRR domain replaced by the A2 domain, whether cleavagealso involves Kuzbanian, the fly ADAM/TACE family protease.Nevertheless, these results provide strong in vivo evidence for amechanical pulling force that is generated by the endocytosis ofDelta in the range required for Notch activation (Langridge andStruhl, 2017).

In sum, studies over the past thirty years have revealed thatNotch mediates cell-cell communication between direct neighborsvia a very simple and direct mechanism from the cell surface tothe genome. This simple mechanism can only operate whencore components (e.g. ADAM/TACE metalloproteases and theγ-secretase) are active. In this regard, we note that the context-specific regulation of these core components has been reported(Dornier et al., 2012; Seegar et al., 2017; Upadhyay et al., 2013).Furthermore, this simple mechanism can be modulated at variouslevels, starting with receptor-ligand interactions that are well knownto be modulated by sugar modifications (Bruckner et al., 2000;Moloney et al., 2000; Okajima and Irvine, 2002; recent reviewby Harvey and Haltiwanger, 2018). Indeed, recent work hasrevealed that fucose modification of EGF8 and EGF12 by Lfngenhances Notch1 binding to and activation by the Notch liganddelta like canonical Notch ligand 1 (Dll1) but not the Notch ligandJag1 (Kakuda and Haltiwanger, 2017; Luca et al., 2017), suggestingnew ways to modulate Notch activation (Schneider et al., 2018).Along the same line, the recent identification of a lipid-bindingdomain in the C2-DSL domain of Notch ligands, which is involvedin Notch binding, suggests that lipids may stabilize ligand-receptorcomplexes and modulate signaling (Suckling et al., 2017). Finally,an alternative ligand-independent mechanism of receptor activationhas also been reported (review by Schnute et al., 2018). It involves

Cell state/fate (output)

Ligand (Delta, Jagged)

Receptor (Notch)

Metalloprotease (ADAM/TACE)

γ-secretase

Effectors (HES)

Transcription activationcomplex (CSL-NICD-Mam)

Processing of the receptoris coupled to its activation

Short lived NICD provides atransient signal to the genome

Ligand down-regulation is coupled to signaling activity

Cell state/fate (input)

Direct signal transduction;no signal amplification

E3 ubiquitin ligase(Neuralized, Mindbomb)

NICD

Fig. 1. An overview of the Notch signaling pathway.Notchmediates contact-dependent signaling between cells. A pairof signal-sending (top) and signal-receiving (bottom) cells isshown here. The ability of a cell to signal depends on its state/fate and varies with ligand identity and expression levels atthe cell surface. It is also modulated by E3 ubiquitin ligases,which can target ligands for endocytosis. Upon activation,Notch is sequentially processed, first by metalloproteases ofthe ADAM/TACE family, then by the γ-secretase, leading tothe release of NICD from the membrane. In the nucleus,NICD assembles with a DNA-binding protein, CSL, and aco-activator, Mam, to form a complex that regulates geneexpression. Among the key target genes and effectors ofNotch are the HES proteins that act in many contexts tomodulate cell state and/or fate. Pathway dynamics arean intrinsic feature of Notch signaling: ligand levels areintimately linked to degradation; receptor processing iscoupled to activation; in the absence of signal amplification,signal transduction is direct; and the response of the genomeis transient, as NICD turns over rapidly.

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the trafficking of full-length Notch to late endosomes and itsprocessing at the limiting membrane of maturing endosomes uponfusion with lysosomes (Schneider et al., 2013). In this context, theshedding of extracellular Notch is thought to be force- and ADAM/TACE-independent. However, this activation mechanism is mostlyobserved when Notch is abnormally stabilized at the limitingmembranes of maturing endosomes upon genetic perturbations, andits contribution to Notch-mediated cell-cell communication inliving organisms appears to be limited.

Cis-inhibition: a simple regulation of general relevanceNotch receptors can also interact with their ligands in cis, i.e. withinthe same cell. Cis- and trans-ligands are thought to compete for

binding to the same region of Notch, but cis-ligands likely failto activate Notch because no cellular force can pull onto theligand/receptor bridge to change the NRR conformation and exposethe S2 site. Thus, cis-ligands inhibit Notch through a non-catalyticprocess that results from simple sequestration of Notch receptors(review by del Álamo et al., 2011). This view is supported bythe recent analysis of orthogonal ligand/receptor pairs in whichthe extracellular domains of Delta/Notch were replaced withheterologous ectodomains: in all cases studied, cis-inhibition wasobserved, arguing that it requires no special property other thancis-binding (Langridge and Struhl, 2017). Thus, cis-inhibition ofNotch appears to be a simple, general and intrinsic property ofNotch signaling.

NeuralizedMindbomb

Ubiquitin

Pulling force

NICDClosed NRR

Open NRR

EGF11-12

S2 S3

EGF8-12

DSL/C2

ADAM γ-secretase

Notch

Delta/Jagged

Step 1: binding

NICDvWF

Wild-type A2 domain

Pulling force: too weakto unfold wild-type A2

vWFMutant A2 domain

FSHR

FSH

A

B

EGF1-3/DSL/C2

Step 2: pulling, catch bond and NRR opening

Step 3: processing

Pulling force: strong enoughto unfold mutant A2

Fig. 2. Receptor activation by mechanical allostery. (A) Receptor activation is initiated (Step 1) through ligand binding, with the EGF repeats 11-12 of a Notchreceptor interacting with the DSL (Delta, Serrate, Lag-1) domain of a Notch ligand. Modification of the intracellular tail of the Notch ligand by the E3 ubiquitinligases Neuralized and/or Mindbomb promotes the endocytosis of the ligands (Step 2). An increase in the interaction surface, which now also involves EGF8-10of Notch and EGF1-3 of the ligand (a ‘catch-bond’ mechanism), allows receptor-ligand interactions to resist the pulling forces that are associated with ligandendocytosis. Pulling forces that are exerted onto Notch mechanically modify the structure of the NRR, converting it from a closed configuration (red) to an openone (green), which renders the S2 site of Notch accessible to ADAM proteases. This ligand-dependent cleavage (Step 3) at the cell surface is then followedby an intra-membrane cleavage at the S3 site by the γ-secretase. This eventually results in the release of NICD. (B) The pulling forces produced by theendocytosis of the chimeric Delta-FSH ligand are too weak to unfold the wild-type A2 domain of the vWF moiety (red spring) that is present in the chimericFSHR-A2-Notch receptor. In contrast, these forces are sufficient to destabilize a mutant version of this A2 domain (green spring), as monitored by the releaseof NICD from this chimeric receptor (Langridge and Struhl, 2017).

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Cis-inhibition may confer specific properties to Notch-baseddecisions. First, cis-ligands can act in vivo as a buffering mechanismthat limits ligand-independent activation of Notch. Second, cis-ligands were proposed to confer ultra-sensitivity to the switchbetween two mutually exclusive signaling states in a cell, the ONstate (low cis-ligand levels, hence trans-activation) and the OFFstate (high cis-ligand levels). This switch allows a sharp transitionbetween the two signaling states (Sprinzak et al., 2010), andmodeling studies have indicated that this property may be relevantfor patterning dynamics (Sprinzak et al., 2011) and signalingprecision (Barad et al., 2010). Finally, cis-inhibition couldcontribute to reducing the strength of Notch signaling bycompeting with trans-ligands for interaction with Notch.Although this prediction appears to be rather obvious, testing itexperimentally in vivo is challenging, as cis- and trans-bindingproperties cannot be easily uncoupled (but see Miller et al., 2009 fora nice exception). Thus, despite its likely broad relevance, thisinteresting regulatory mechanism is most often overlooked. In thisregard, developing tools to differentially detect Notch in an inactive/auto-inhibited, cis-inhibited or trans-interacting state would greatlyadvance the analysis of this general regulatory mechanism.

Cell-cell contact and signaling sitesAs mentioned above, ligand endocytosis is generally required forreceptor activation; it is thought to provide the necessary force tounfold the NRR in vivo. This in turn implies that S2 cleavage usuallytakes place at the cell surface. Thus, ligand-receptor trans-interaction, ligand endocytosis and S2 cleavage all likely co-occurat the cell surface. Various cell-cell contact sites at which theseprocesses might occur have been proposed. These range from apicalcell junctions in epithelia (Hatakeyama et al., 2014; Sasaki et al.,2007) to apical cell membranes (Lopez-Schier and Johnston, 2001),basal protrusions (Cohen et al., 2010) and/or long and specializedcellular extensions (Eom et al., 2015). However, in none of thesecases is the exact location of Notch receptor activation defined.Determining where receptor activation takes place at the subcellularlevel is relevant for three main reasons. First, co-localization ofligands and receptors does not predict signaling, as cis-interactionsmight dominate. Second, the size and shape of these signaling cell-cell contacts might regulate signaling strength (Shaya et al., 2017).Third, the spatial range of signaling may be extended through longcellular protrusions that reach beyond immediate neighbors, as hasbeen proposed for long cellular extensions in zebrafish (Eom et al.,2015) and basal filopodia in Drosophila (Cohen et al., 2010).Recently, a new approach has been developed and applied to

determine the membrane domain at which Notch is activated(Trylinski et al., 2017). This method relies on receptors that areintracellularly tagged by fluorescent proteins such that NICD can bedetected in the nucleus. To determine where NICD comes from, thefluorescence properties of Notch at a particular cell surface site canbe specifically modified, e.g. by photo-bleaching or photo-conversion, allowing receptor activation to be monitored bytracking the nuclear accumulation of modified NICD fluorescenceover time. This approach was applied in the context of a binary fatedecision that immediately follows asymmetric division of neuralprecursor cells in Drosophila. It revealed that only a specific subsetof Notch receptors, located basal to the midbody, contribute toNICD release in this context (Trylinski et al., 2017). Restrictingsignaling to this new cell-cell interface may be key for intra-lineagefate decisions. Given the importance of membrane trafficking inNotch signaling, it is tempting to speculate that the regulatedtargeting of Notch to specific cell-cell contacts at which signaling

takes place may, in some cases, underlie the functional requirementfor membrane trafficking in Notch signaling.

Of note, although this method can identify signaling pools ofNotch at the membrane, it does not directly identify where S2and S3 cleavage take place. Indeed, S3 cleavage may occur atboth the plasma membrane and at the limiting membrane ofendomembranes, following S2 cleavage and internalization of themembrane-tethered form of intracellular Notch (Vaccari et al., 2008;Narui and Salaita, 2013; Sorensen and Conner, 2010). Thistrafficking step introduces a time delay and allows for furtherregulation. In addition, the cellular location of S3 cleavage appearsto also have an impact on the precise peptide bond that is cleaved bythe γ-secretase, resulting in NICD fragments with distinct amino-termini. Indeed, cleavage at the cell surface was reported to producea moderately stable NICD with an N-terminal valine, whereascleavage at the limiting membrane of endosomes produces short-lived NICD (Tagami et al., 2008) owing to the N-end rule proteindegradation pathway (Varshavsky, 2011). Signaling strength mightalso be downmodulated by targeting S2-cleaved receptors fordegradation before S3 cleavage. Therefore, further analysis of thestate of Notch at signaling sites is required and would benefit fromnew tools that could be used to monitor and locally manipulateforce-dependent S2 cleavage and further processing by γ-secretase.

Notch signaling and tissue mechanicsRecent studies have highlighted the interplay between cell-cellsignaling and tissue mechanics in pattern formation andorganogenesis (Shyer et al., 2017). As a ligand-dependentmechanosensitive receptor, Notch integrates mechanical andchemical/molecular cues. However, an open question is whetherNotch integrates molecular cues (Notch ligands) with force acting atthe supracellular scale, and not merely at the subcellular scale, as isthe case during endocytosis. For example, could it be that shearstress along cell-cell contacts contributes to ligand-dependentreceptor activation, possibly by lowering the force threshold formechanical allostery? The observation that mechanical stressgenerated by the high-mobility of Notch-expressing T-cells on aplastic surface that is covered with immobile ligands leads toreceptor activation is, at least in this artificial context, consistentwith this possibility (Varnum-Finney et al., 2000). Similarly,ligands at the surface of a supported lipid membrane surface aremore active when their lateral mobility is restricted (Narui andSalaita, 2013). Moreover, force-induced destabilization of the NRR(Notch activation) may not necessarily require the force to beinduced by ligand binding. For example, EDTA treatment appearsto destabilize the NRR of Notch in Drosophila S2 cells, whichresults in ligand-independent S2 cleavage (Rand et al., 2000). Inaddition, physiological ligand-independent activation of Notchthrough S2 cleavage has recently been reported in the Drosophilafollicular epithelium (Palmer et al., 2014) and in mammalian T-cells(Steinbuck et al., 2018). Of note, these signaling events differ fromthose that result from the mis-trafficking of Notch towards lateendosomal compartments, in which it is proteolytically cleaved by amechanism that does not involve S2 cleavage and may not involveforce (Schneider et al., 2013). How the S2 site becomes unmaskedin a ligand-independent manner in fly follicular cells and in mouseT-cells is still unknown and it will be interesting to decipher whetherand how mechanics also contribute to the destabilization of theNRR in these contexts.

Interestingly, Notch signaling is required in adult blood vessels tomaintain endothelial cell barrier integrity in response to flow/hemodynamic fluid stress (Lagendijk et al., 2018), and it has also

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been shown that shear stress is required for the activation of Notch1in endothelial cells in vitro, raising the possibility that blood flowcontributes directly to receptor activation (Fang et al., 2017;Loerakker et al., 2018). Alternatively, it is possible that shear stressindirectly regulates Notch signaling, for example via theendocytosis of delta like canonical Notch ligand 4 (Dll4), theligand that activates Notch1 in endothelial cells through mechanicaleffects on the endothelial cell cytoskeleton. Whether forces exertedby fluid shear stress on Notch1 receptors at endothelial cellmembranes contribute to NRR unfolding remain to be tested.Whatever the role of shear stress on Notch1 signaling, recent in vitrowork points to an additional mechanism throughwhich Notch mighthelp strengthen the endothelial cell barrier in response to fluid shearstress, without involving its transcriptional activity (Polacheck et al.,2017). This mechanism is mediated by a fragment of the Notch1receptor that includes its transmembrane domain (TMD) and that isproduced following Dll4-dependent S2 and S3 cleavages andrelease of the extracellular domain (ECD) and NICD fragments. Thepredicted TMD fragment is proposed to promote the assembly of acomplex in the plasma membrane that consists of VE-cadherin(cadherin 5), the phosphatase Lar and the Rac1 guanidine exchangefactor trio; this complex triggers an increase in Rac1 activity tostabilize adherens junctions and promote barrier integrity(Polacheck et al., 2017). However, the predicted TMD fragmentthat is proposed to interact with VE-cadherin was shown in previouswork to produce, following further intramembrane cleavage,secreted extracellular Notch Aβ like-peptides (Nβ) (Okochi et al.,2006), which implies that assembly of this complex must beremarkably efficient to compete with release of the Nβ peptide. Inparallel to this proposed non-transcriptional mechanism, flow andshear stress in the same microvasculature model result in NICD-mediated induction of target genes such as Hey1 and Hes1. It willtherefore be important to determine the relative contribution of thesetwo mechanisms to the response of endothelial cells to flow-mediated shear stress. Thus, whereas Notch1 senses force andregulates tissue integrity in endothelia (and possibly other tissues),the extent to which chemical/molecular and mechanical cues areintegrated at the level of the receptor itself or up- or downstream ofreceptor activation awaits further analysis.

Dynamic encoding at the cell surfaceNotch receptors have more than one ligand. For example, twoligands (Delta and Serrate) are found inDrosophila and five (DLL1,DLL3, DLL4, JAG1 and JAG2) are found in humans. The existenceof multiple ligands accounts, in part, for the complex regulation ofNotch activity in time and space, and may also contribute tomodulating signal levels and duration. For example, signaling by astrong trans-ligand can be reduced upon expression of a secondweaker trans-ligand, as the latter competes for binding to Notch(Benedito et al., 2009; Petrovic et al., 2014). Perhaps less intuitively,different ligands activating the same receptor may trigger differentresponses at the genome level. For example, Notch1 activation byDll1 in the myotome of chick embryos leads to strong Hes1expression, whereas activation by Dll4 results in Hey1 expression,with opposite effects on myogenesis (Nandagopal et al., 2018). Asthese different ligands have a single and shared primary output, i.e.NICD, it is not clear how the information associated with ligandidentity is transduced by NICD. A recent study (Nandagopal et al.,2018) provides a solution to this problem and suggests thatinformation is encoded through distinct signaling dynamics(Nandagopal et al., 2018; Purvis and Lahav, 2013). A first hint atthis solution comes from the observation that Hes1 and Hey1 genes

respond differently to Notch dynamics in cultured cells that havebeen engineered to conditionally express a constitutively activeform of Notch (Nandagopal et al., 2018). Specifically, a short pulseof Notch activation is sufficient to induce a transient peak of Hes1expression, the amplitude of which does not significantly vary withthe duration of the pulse. However, sustained Notch activation leadsnot only toHes1 expression but also toHey1 expression. Activationof different genes by Dll1 and Dll4 might thus derive from distinctdynamics of NICD production caused by each ligand. To test thishypothesis, the NICD production rate was calculated at the singlecell level in an ex vivo assay, by measuring the output of a GAL4-driven promoter coupled to a stable fluorescent reporter, in responseto a ligand-activated chimeric Notch1-Gal4 receptor (Nandagopalet al., 2018). By design, this system preserves Notch activationdynamics, allows signal amplification to facilitate detection andprevents cross-talk with the endogenous signal transductionmachinery. ‘Receiver’ cells were cultured together with ‘sender’cells, and the results revealed that when these cells express Dll4, theactivity of the NICD-responsive promoter increases steadily to reacha plateau, the level of which depends on Dll4 levels (Fig. 3A). Thus,Notch is activated in a sustained manner by Dll4, and signalingstrength depends on Dll4 levels. By contrast, discrete and transientpulses of promoter activity are observed when ‘receiver’ cells are incontact with Dll1-expressing cells (Fig. 3A). These pulses wereestimated to last ∼1 h, and their frequency, but not amplitude,depends on the levels of Dll1. Thus, in this ex vivo assay, Dll1 levelsregulate pulse frequency whereas Dll4 levels modulate theamplitude of the response (Nandagopal et al., 2018). Interestingly,this difference in Notch dynamics elicited by Dll1 or Dll4 maps totheir intracellular domains (ICD): whereas a chimeric ligandcontaining Dll1 ICD linked to the ECD of Dll4 [Dll4(ECD)-Dll1(ICD)] produces a pulsatile Notch activity, the reverseDll1(ECD)-Dll4(ICD) chimera generates a sustained Notchresponse, as with Dll4 itself (Nandagopal et al., 2018).Furthermore, how each ICD confers the observed ligand-specificdynamic activation may be related to its ability to regulate trans-endocytosis of Notch: Dll1 appears to trigger trans-endocytosiswhen Notch receptors are clustered, possibly resulting in pulses ofNICD production, whereas Dll4 triggers signaling from dispersedNotch receptors, possibly resulting in a continuous, steady NICDproduction rate (Nandagopal et al., 2018). Whether the ICDs of Dll1and Dll4 interact with different molecules that contribute to thesedistinct capacities is a possibility that remains to be explored. Wenote, however, that although Dll1 function cannot be replaced byDll4 in mesodermal tissues of the mouse embryo, the ICD of Dll1can be functionally substituted by the ICD of Dll4 in the sametissues (Tveriakhina et al., 2018). This suggests that a possiblealteration in Notch dynamics by the Dll4 ICD (which causes asustained NICD production) might be buffered in vivo by othercomponents of the Notch response. It would therefore be importantto monitor signaling dynamics in vivo and correlate them with thephenotypic consequences of altering ligand structures. In addition,as many tissues express more than one receptor as well as more thanone ligand, it will be important to study the response dynamics ofdifferent Notch receptors to their various ligands, and to investigatehow dynamic encoding is employed in a combinatorial mannerin vivo.

Decoding Notch dynamics in the nucleusThe findings above identify a novel mechanism – based on temporaldynamics of receptor activation at the cell surface leading to distinctrates of NICD production – through which Notch transduces

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information from ligand-expressing cells to the nucleus. However,how distinct rates of NICD production are mechanistically decodedat the genome level to direct expression of distinct sets of Notchtargets remains to be studied in vivo. In the case of Hes1 and Hey1,their different expression profiles in response to Notch activation bydifferent ligands may, in principle, involve a downstream circuitryof auto- and cross-inhibition among these genes (Fig. 3B,C), similarto that observed for other genes of this family (Fior and Henrique,2005; Schröter et al., 2012; Trofka et al., 2012). We speculate that,although both genes are initially upregulated following Notchactivation, Hes1 accumulates faster than Hey1, for example becauseof optimal CSL/NICD-binding sites in the Hes1 promoter,repressing Hey1 gene expression. Then, as Hes1 levels decreasebecause of protein instability and auto-repression (Hirata et al.,2002), two temporal patterns of Hey1 expression could emerge. Inthe case of Notch activation by Dll1, the proposed bursts of NICDand Hes1 production are coupled such that Hes1 keeps Hey1repressed whenever NICD is induced. However, when Notch isactivated by Dll4, sustained NICD production allows for Hey1expression when Hes1 levels drop, resulting in a delayed wave ofHey1 gene activation (Fig. 3C). It will be of interest to study theextent to which a regulatory circuit such as this underlies thecontrasting response of Notch targets to different ligands.In addition to the information that is encoded in Notch ligands,

each responding cell is predicted to display a set of accessible Notch-responsive elements (NREs) that allow it to mount a specifictranscriptional program following Notch activation. NREaccessibility likely depends on the state of the cell, which itself isinfluenced by the local environment and developmental history of thecell. Several studies have attempted to define the chromatinenvironment that is associated with open or closed NREs in various

cell types by mapping chromatin conformation and modificationsin relation to the presence of CSL and NICD. For example, inDrosophila cell lines, as well as in mammalian C2C12 cells andT-lymphoblastic leukemia (T-LL) cell lines, CSL-bound loci arepreferentially enriched in a particular array of histone modifications(H3K4me1, H3K27ac and H3K56ac), but this combination alone isnot enough to predict which of the CSL-bound sites are capable ofrecruiting NICD (Skalska et al., 2015; Wang et al., 2011). Otherfactors are therefore required to establish the appropriate regulatoryenvironment that allows specific NREs to become competent forCSL/NICD binding. By searching for transcription factor (TF)-binding sites that appear to be frequently associated with NREs,various candidate TFs that might play crucial roles in priming NREsfor enhancer activity, in a lineage-specific manner, have beenidentified. A good example is the Drosophila Runt-domain TFLozenge, which is required to make NREs competent for Notchactivation in fly hemocytes (Terriente-Felix et al., 2013). Bindingsites for Runx1, the mammalian homologue of Lozenge, were alsofound to be enriched close to CSL-Notch1 binding sites, both in T-LLcells and in the mouse kidney cell line mK4 (Wang et al., 2014; Hasset al., 2015). It was further shown that RUNX1 cooperates withNOTCH1 in activating the expression of the target gene IL7R throughan enhancer that contains adjacent CSL and RUNX1 binding sites(Wang et al., 2014). These examples are likely just the tip of theiceberg, and there may be other TFs that cooperatewith CSL-NICD ina lineage-specific manner to regulate which genes are competent torespond to Notch activation. Further work is therefore needed toidentify these TFs and understand how their interaction withCSL-NICD regulates target gene accessibility and responsiveness.

Another regulatory layer involves the different sensitivities toNICD levels that are displayed by each individual NRE, which vary

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Fig. 3. Encoding and decoding ligand information. (A) Different dynamics of NICD production are triggered by Dll1 or Dll4. When Notch1-receiver cells(green) are exposed to Dll1-expressing cells (orange, left), pulses of NICD release are observed, the frequency of which depends on Dll1 expression levels.In contrast, signaling from Dll4-expressing cells (pink, right) is able to sustain the continuous production of NICD, the levels of which correlate with Dll4 levels(Nandagopal et al., 2018). (B) The regulatory circuit proposed to decode ligand information applied to the Hes1 and Hey1 genes is shown. NICD levels areproposed to be interpreted differently by the Hes1 and Hey1 promoters, resulting in faster increase of Hes1 transcription and Hes1 accumulation. In this circuit,Hes1 inhibits its own expression and also represses Hey1. We suggest that high Hes1 levels repress NICD-driven Hey1 transcription, until a point when Hes1levels start to decay, because of negative feedback and protein instability. At this point, Hey1 transcription would raise and Hey1 would start to accumulate.Additional repression of Hes1 transcription by Hey1 might indirectly contribute to increased Hey1 transcription. (C) The proposed regulatory circuit outlined inpanel B would generate different responses in receiving cells, according to the ligand (Dll1 or Dll4) to which they are exposed. In cells exposed to high levels ofDll1 (left), NICD pulses (light blue) are likely to generate similar pulses of Hes1 (red). If these pulses occur frequently, Hes1 levels will never decay to a levelthat would allowHey1 de-repression. Therefore, a strong Dll1 signal may not result inHey1 activation (dark blue). On the contrary, sustained levels of NICD drivenby high levels of Dll4 (right) would result in Hey1 expression (dark blue). After the initial burst of Hes1 transcription (red), Hes1 auto-repression and fast proteindecay might reduce Hes1 levels below a threshold allowing for Hey1 de-repression, such that sustained NICD (light blue) would activate Hey1 expression.

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according to the number, type and affinity of CSL-binding sites thateach NRE contains. These sites can occur as single sites or pairedsites, known as Su(H)-Paired Sites (SPS), which consist of twohigh-affinity binding sites that are separated by 15-17 base pairs andare found in a head-to-head orientation (Bailey and Posakony, 1995;Ong et al., 2006). Diverse combinations of CSL-binding sites can befound in individual NREs and these, together with neighboring TF-binding sites, are likely to define unique regulatory architectures foreach Notch target gene that allow it to decode nuclear NICD levelsand modulate its transcriptional activity accordingly (Seversonet al., 2017). Assuming rapid and commensurate kinetics for bothreceptor activation/NICD production and NICD degradation,varying nuclear NICD levels may reflect different rates of NICDproduction, which, as described above, can be elicited by distinctligands. Thus, the challenge is to understand how the dynamicinteraction between CSL, NICD and associated proteins canmechanistically interpret the information that is received fromneighboring cells. In the current view, when signaling is inoperativeand NICD is not produced, CSL dynamically binds its target DNAsites and recruits a repressive complex that maintains Notch targetsin a silenced state. When Notch is active and NICD levels rise in thenucleus, a switch from repression to activation is proposed to occurat this subset of ‘open’ Notch targets. Two possible mechanismsmay underlie this switch. First, NICD may interact with DNA-bound CSL and displace the corepressors while recruitingcoactivators. This view is, however, difficult to reconcile with thefinding that corepressors and NICD have comparable high affinitiesfor CSL (Oswald and Kovall, 2018), making a competitivedisplacement mechanism unlikely to generate robust and sensitiveresponses. Alternatively, NICD may assemble an activationcomplex outside of DNA and this complex may dynamicallycompete with repression complexes for target sites. To test these twopossibilities, a recent fluorescence recovery after photobleaching-based study examined the dynamic behavior of CSL in vivo, both inrepressive conditions (Notch-OFF) and in the presence of NICD(Notch-ON), using Drosophila salivary glands as a cellular system(Gomez-Lamarca et al., 2018). The analysis of CSL in Notch-OFFconditions revealed that it exhibits fast dynamics in the nucleus:only about one third of CSL molecules are predicted to be bound ateach time to DNA, showing a very short residence time of 0.5-2 s.Upon constitutive expression of NICD (Notch-ON), CSL retainsits fast dynamics globally, but is seen to accumulate at a specificlocation on polytene chromosomes that corresponds to the E(spl)-Clocus, which contains a cluster of Notch target genes with multiplehigh-affinity CSL-binding sites. This accumulation reflects anincreased residence time (of up to 10-15 s) of CSL at E(spl)-C, andis indeed dependent on the interaction of CSL with NICD; notably,a mutant CSL that is unable to bind NICD exhibits a shorterresidence time and shows no visible accumulation at the E(spl)-Clocus (Gomez-Lamarca et al., 2018). It was further shown that theCSL/NICD complex establishes a more open chromatinenvironment at the E(spl)-C locus, as measured by the ATACmethod as well as by the increased detection of H3K27Ac andH3K4me1, which are two histone modifications normallyassociated with open, active enhancers. The increase in H3K27Acis likely mediated by recruitment of the histone acetyltransferaseCBP/p300 to the CSL/NICD complex, whereas the H3K4 mono-methylase KMTD2 (Trithorax-related, Trr, in Drosophila) wasshown to be responsible for the observed increase in chromatinaccessibility in Notch-ON cells (Gomez-Lamarca et al., 2018).Strikingly, in Notch-ON cells depleted for Trr, no CSLaccumulation at the E(spl)-C locus is observed, and the locus

remains silent. This indicates that Trr is required to modifychromatin at Notch target genes and promote transcriptionalactivation by NICD. As KMTD2 interacts with a CSL corepressorin mammals (Oswald et al., 2016), it appears that the CSL repressorcomplex might prime Notch targets for NICD activation.Conversely, the increased chromatin accessibility promoted by theCSL-NICD complex appears to also facilitate access of the CSLcorepressor complex to Notch targets. Such an ‘assisted’ loadingmechanism might therefore allow NICD to indirectly stimulatechromatin opening and boost transcriptional activity (Gomez-Lamarca et al., 2018).

In summary, the observed dynamic turnover of CSL and NICD attarget sites suggests that CSL-NICD occupancy levels should behighly sensitive to NICD concentration in the nucleus. Indeed, shortresidence times imply that high local concentrations of NICD andCSL at target genes have to be attained in order to extend occupancyfor enough time to recruit other components and assemble afunctional, transcriptionally active complex. The dynamic CSL hubthus provides a system that may be tunable and responsive tofluctuations in NICD concentrations. This might account for theobserved heterogeneity in transcriptional activation of target genesin Notch-ON cells (Lee et al., 2016), in which some of these genesare reported to be asynchronously activated within the same cell,possibly reflecting the dynamic turnover of CSL complexes and theinherent fluctuations in NICD concentration at these targets.

Notch in context: distinct outcomes within a single lineageGiven the relative simplicity of the core Notch signaling mechanism,it is remarkable how this cell-cell communication pathwayparticipates in so many diverse cell fate decisions. A simple andstriking example of this versatility is provided by the intestinal stemcell (ISC) lineage in the Drosophila midgut epithelium (Micchelliand Perrimon, 2006; Ohlstein and Spradling, 2006). In this lineage,multipotent ISCs divide to self-renew and to produce a transientprogenitor that either differentiates into an absorptive enterocyte (EC)or divides once to produce two secretory enteroendocrine (EE) cells(Chen et al., 2018; Guo and Ohlstein, 2015) (Fig. 4A). Remarkably,Notch is required for both ISC-EC and ISC-EE fate decisions(Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006), butsignaling acts in opposite directions in the two decisions, relative to inthe self-renewed ISC (Guo and Ohlstein, 2015). In ISC divisions thatproduce an EC, Delta signals from the newly forming ISC andactivates Notch in the sister cell that will become an EC. On thecontrary, when an ISC divides to produce an EE progenitor, Deltasignals from this progenitor to activate Notch in the sister cell that willbecome a new ISC (Fig. 4B). Thus, the sameDelta signal is employedin different settings within the same lineage to regulate either ECdifferentiation or ISC self-renewal.

How this occurs is unclear. One possibility is that these twolineage decisions may involve different strengths of Notch activity,and the Notch antagonist Numb may be one of the factors requiredto modulate the Notch response in the ISC-EE decision (Sallé et al.,2017). Context is therefore key to explain howNotch regulates thesetwo seemingly opposite outcomes. In addition, recent worksuggests that ISCs fluctuate between two states, associated withdistinct levels of the pro-EE transcription factor Scute (Chen et al.,2018). When an ISC reaches high Scute levels, it starts expressingProspero (Pros), a downstream target of Scute that is required toimplement an EE fate. At division, Pros is proposed to be unequallysegregated into the daughter cell that will become an EE progenitor(Guo and Ohlstein, 2015). In this context, Delta is expressed in theEE progenitor and the signal-receiving cell is the self-renewed ISC,

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in which Notch activity is required to switch off the pro-EE factorScute and thereby prevent EE differentiation. These low Scute andPros-negative ISCs may then undergo several rounds of divisions,each producing a self-renewed ISC and an EC progenitor (Fig. 4B,dashed arrow). In this context, Notch signaling also becomesdirectional, with Delta-expressing ISCs activating Notch in ECprogenitors (Micchelli and Perrimon, 2006; Ohlstein and Spradling,

2006). Here, Notch activity may function both to inhibit the pro-EEdeterminant Scute and to antagonize the transcription factorDaughterless, which has previously been shown to be required tomaintain ISC identity (Bardin et al., 2010). We therefore suggestthat a common and key function of Delta/Notch signaling in thislineage is to restrain the dynamic expression of Scute in ISCs, likelythrough repressor E(spl) proteins. These Notch targets were shownto also be induced by Scute in ISCs, and it has been proposed thatreciprocal regulation between Scute and E(spl) may keep Scuteexpression at low levels in most ISCs (Chen et al., 2018). However,when Scute autoregulation becomes active in a given ISC, thiscauses a switch to a high-Scute state, leading to the production ofEEs, before reverting back to a low Scute state upon Notchactivation. Thus, the interplay of Notch with Scute dynamics in theISC lineage may provide a simple mechanism to understand howtwo seemingly distinct cell fate decisions can be governed by asingle molecular process. As other stem cell lineages may follow thesame lineage logic, e.g. the airway epithelium (Pardo-Saganta et al.,2015) in which Notch regulates a Scute-related factor (Borges et al.,1997;Mori et al., 2015; Rock et al., 2011), one may wonder whetherfate decisions seen as distinct might be explained by a unifiedmechanism as Notch dynamics become unraveled.

Self-organized Notch dynamics in patterningA classic function of Notch is to single-out individual cells fromgroups of cells with similar developmental potential via a processtermed ‘lateral inhibition’. In this process, the selected cells send aninhibitory signal to prevent other cells in the group from adopting thesame fate. Lateral inhibition can operate in time to limit the number ofcells adopting a given fate in response to a general inductive signal,thereby maintaining a pool of uncommitted progenitors (Chitniset al., 1995). It can also operate in space to mediate patterning viathe differentiation of regularly spaced cells (Simpson, 1990). Forexample, during peripheral neurogenesis in flies, Notch signaling isknown to play a role in selecting sensory organ precursor cells (SOPs)fromgroups of equipotent cells that are endowed to become neural viathe expression of proneural factors (Simpson, 1990). This occurs aspart of a two-step process (Ghysen and Dambly-Chaudiere, 1989).First, in response to developmental signals that convey positional andtemporal information, proneural factors are expressed in a restrictedmanner within the neuroepithelium. Second, Notch-mediatedinhibitory cell-cell interactions single-out a limited number ofregularly spaced neural progenitors from these proneural clusters.This two-step model also applies to the formation of the largemechanosensory organs (macrochaetae) that are found at stereotypedpositions on the Drosophila thorax (Gómez-Skarmeta et al., 2003).

In contrast with this two-step model, a recent study on thepatterning of smaller microchaetae in Drosophila suggests thatNotch acts in a continuous manner to both establish proneuraldomains and select neural progenitors in the dorsal-central region ofthe pupal notum (Corson et al., 2017). In this context, Notch appearsto regulate what is usually seen as two distinct steps in sensory organformation (i.e. establishment of proneural clusters and selection ofneural progenitors with these clusters) via a single developmentaloperation through its interplay with proneural factor dynamics.Briefly, a stable activity pattern of Notch at the onset of patterningprovides a negative template to define where proneural genes can beswitched on at metamorphosis, such that the two stripes of cells withinitially high Notch activity become flanked by three proneuralstripes. Then, as the ability of cells to produce and respond to aNotch signal is modulated by proneural factors, the pattern of Notchactivity dynamically evolves. These dynamic changes define two

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Fig. 4. A unified view of Notch signaling in Drosophila ISCs. (A) In theDrosophila midgut epithelium, multipotent ISCs divide to self-renew and toproduce a progenitor that either differentiates into an absorptive enterocyte(EC) or divides once to produce two secretory enteroendocrine (EE) cells.Early studies suggested that high Delta signaling from the ISC is requiredfor EC differentiation, whereas weak Delta activity favors EE differentiation(Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006). (B) ISCs areproposed to fluctuate between two states of Scute expression, high and low(Chen et al., 2018). ISCs in a high Scute state (pink) divide to give rise toan enteroendocrine progenitor (EEp) and another ISC. Delta from the EEp thenactivates Notch in the sister ISC (green nucleus) to repress Scute expressionand reset its potential: this cell returns to a low Scute state (pale orange).The EEp divides once to produce two EEs. At the low Scute state, ISCs candivide to give rise to a committed enteroblast (EB; or enterocyte progenitor,ECp), and another ISC. Delta from the self-renewed ISC activates Notch in thesister ECp (green nucleus), in which it may act to prevent Scute activationand EEp commitment. Therefore, we propose that a common function ofNotch activity is to inhibit the activity of Scute, independent of whether theISC is the signal-sending cell (low Scute state) or the signal-receiving cell(high Scute state).

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new regions of cells with low Notch activity, intercalated betweenexisting proneural stripes, to produce two additional proneuralstripes. This self-organized process therefore creates a pattern of fiveproneural stripes. Each of these stripes then progressively resolves toproduce singled-out SOPs, eventually giving rise to a final pattern offive rows of sensory bristles (Corson et al., 2017). The geometry anddynamics of this patterning process can be faithfully produced in asimple mathematical model based on bistability that involves asingle variable describing the state of cell, when given appropriateinitial and boundary conditions (Corson et al., 2017). Remarkably,both stripe patterning and the resolution of proneural stripes intoregularly spaced neural progenitors are the sequential outputs of asingle logical operation with a unique set of parameters (Fig. 5).Thus, modeling suggests that what is often viewed as a two-stepprocess, with first the specification of proneural clusters and then thesingling out of neural progenitors, may actually involve a singlecontinuous self-organized process. This therefore strongly suggeststhat signaling by Notch can mediate both proneural patterning andSOP selection via a single logical operation deployed in time andspace to robustly produce a complex stereotyped pattern (Corsonet al., 2017). Whether Notch-mediated self-organization similarlyoperates at the tissue scale to regulate the patterning of otherdifferentiated cells is not yet known. We note, however, that theconstraints and assumptions of the mathematical model are generic,suggesting that this model may be of general relevance. Again, thisillustrates how versatility in Notch function in vivo may notnecessarily arise from the complex and context-dependentintegration of Notch signaling with other developmental signalsbut may, in part, arise from self-organized dynamics.

Conclusions and future directionsNotch receptors relay information from the cell surface to thegenome in a simple, linear and stoichiometric manner. At the core ofthe activation mechanism is the ligand- and force-dependent changein the structure of the NRR, which allows for the processing of thereceptor, and the subsequent release of the intracellular domain fromthe membrane. Although we have witnessed impressive recentprogress in our understanding of Notch signaling mechanisms, keychallenges remain. For example, the 3D architecture of the completeextracellular ligand-receptor complex remains to be determined.Recent advances in determining the structure of large protein

complexes at the cell surface, using Cryo-Electron TransmissionMicroscopy, may help to address this challenge. In addition,visualizing receptor activation in real-time, possibly in associationwith ligand endocytosis, and determining the organization ofsignaling trans-complexes, discriminating them from inactive cis-complexes, are important challenges that remain to be addressed.Interestingly, the central signal processing region of the receptor isflanked by an ECD with signal input activity and an ICD that hasoutput activity, and this modularity in design makes it possible toswap input/output functions (Gordon et al., 2015; Lecourtois andSchweisguth, 1998; Struhl and Adachi, 1998; Vooijs et al., 2007).This approach was recently used to build synthetic cell-cellcommunication circuits using synthetic Notch receptors(synNotch) in which both the input and output domains werereplaced with heterologous protein domains, keeping only themechanosensitive and cleavable part of Notch (Morsut et al., 2016).The resulting novel cell-cell signaling devices can thus functionorthogonally to Notch as well as to one another. By combiningdifferent input/output functions, user-defined responses could beobtained to produce diverse multicellular patterns (Morsut et al.,2016). As noted by the authors, however, these synNotch receptorsappear to function in the absence of any endocytic signal,suggesting that endocytosis is unlikely to provide the force torender the S2 site accessible for cleavage (Morsut et al., 2016).Thus, the ligand-dependent mechanism responsible for displacingthe NRR of synNotch is not clear. It is possible that shear stresscontributes to synNotch activation, in which case these synNotchreceptors might become very useful tools to report on shear stressand tissue mechanics in living organisms. More generally, thisapproach opens up a wide range of possible applications formulticellular synthetic biology (Roybal et al., 2016a,b; Toda et al.,2018). Along a similar line, light has emerged as a powerful tool formanipulating the activity and/or localization of target proteins usinglight-sensitive domains. The rational design of light-regulatedligands and/or receptors will be of considerable interest to controlthe timing, pattern and strength of signaling. Combined with recentadvances in CRIPSR-based genome engineering, it should bepossible to move beyond the tagging of endogenous molecules(Corson et al., 2017; Trylinski et al., 2017) and towardsmanipulating the dynamics of endogenous Notch pathwaycomponents with light. The future should be bright!

Initial patternof Notch activity

A first set of proneural stripesat the onset of proneural activity

Stripe resolution into SOPsand emergence of asecond set of stripes

Final pattern of rows

Fig. 5. A single self-organized process patterns proneural stripes and selects progenitors. Snapshots from a simulation (Corson et al., 2017) that mimicsthe dynamic pattern of Notch and proneural activities in the Drosophila pupal notum. These snapshots illustrate the progressive emergence of five rows ofprogenitor cells (magenta), starting from an initial pattern of three stripes of cells with low levels of Notch activity (green). This initial pattern of Notch activity forms anegative template to first produce a complementary pattern of three proneural stripes of cells with intermediate/high levels of Notch activity (orange/red). Asprogenitors emerge fromwithin these first proneural stripes, and as the pattern of Notch dynamically evolves, two new additional stripes intercalate to produce thefinal pattern. Key parameters of this model are: bistability (with two stable cell states: a high proneural activity and low inhibitory signal state, SOP fate, magenta;and a low proneural activity and high inhibitory signal state, non-SOP fate, green); non-linear production of the inhibitory signal in response to proneural activity;and inhibitory signaling beyond immediate neighbors.

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AcknowledgementsWe thank A. Bardin, K. Storey, M. Trylinski and our anonymous referees for usefulcomments and suggestions. We thank F. Corson for Fig. 5.

Competing interestsThe authors declare no competing or financial interests.

FundingOur research received specific grants from the Agence Nationale de la Recherche(ANR16-CE13-0003-02 and ANR-10-LABX-0073) and Fondation pour laRecherche Medicale (DEQ201180339219) to F.S.

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