Author’s Accepted Manuscript Do as I say, Not(ch) as I do: lateral control of cell fate Marika Sjöqvist, Emma R Andersson

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To appear in: Developmental Biology Received date: 15 July 2017 Revised date: 15 September 2017 Accepted date: 26 September 2017 Cite this article as: Marika Sjöqvist and Emma R Andersson, Do as I say, Not(ch) as I do: lateral control of cell fate, Developmental Biology, https://doi.org/10.1016/j.ydbio.2017.09.032 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Do as I say, Not(ch) as I do: lateral control of cell fate Marika Sjöqvist and Emma R Andersson

Department of Biosciences and Nutrition, Karolinska Institutet Correspondence: [email protected]

Keywords: Notch, lateral inhibition, lateral induction, Jagged, Jag1, Jag2, Delta, Dll, Dll1, inner ear, organ of Corti, sensory organ, SOP, patterning, Turing,

ABSTRACT

Breaking symmetry in populations of uniform cells, to induce adoption of an alternative cell fate, is an essential developmental mechanism. Similarly, domain and boundary establishment are crucial steps to forming organs during development. Notch signaling is a pathway ideally suited to mediating precise patterning cues, as both receptors and ligands are membrane-bound and can thus act as a precise switch to toggle cell fates on or off. Fine-tuning of signaling by positive or negative feedback mechanisms dictate whether signaling results in lateral induction or lateral inhibition, respectively, allowing Notch to either induce entire regions of cell specification, or dictate binary fate choices. Furthermore, pathway activity is modulated by Fringe modification of

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receptors or ligands, co-expression of receptors with ligands, mode of ligand presentation, and cell surface area in contact. In this review, we describe how Notch signaling is fine-tuned to mediate lateral induction or lateral inhibition cues, and discuss examples from C.elegans, D. melanogaster and M. musculus. Identifying the cellular machinery dictating the choice between lateral induction and lateral inhibition highlights the versatility of the Notch signaling pathway in development.

INTRODUCTION The development of multicellular organisms, with compartmentalized regions for different functions, such as organs, requires specific patterning cues. Similarly, determining the localization of where a specific cell type should develop relies on precisely regulated patterning of cell populations. Several mechanisms have been proposed to break symmetry and establish patterns for formation of specific structures, including Alan Turings morphogen reaction-diffusion model (Turing, 1952), discussed elsewhere in this issue (Iber, this issue) and positional information, first proposed by Lewis Wolpert (Wolpert, 1969). Interestingly, when Alan Turing first coined the term “morphogen” he defined a morphogen simply as a “form producer”, which with today’s insight into mechanisms driving morphogenesis would encompass an array of “form producers”, from soluble or membrane-bound ligands to secreted exosomes. The reactiondiffusion model however, proposed a mechanism by which chemicals diffusing due to Brownian motion would establish patterns, thus morphogens have typically been thought of as diffusible, even though Alfred Gierer and Hans Meinhardt pointed out that morphogen spreading could be achieved by mechanisms other than molecular diffusion (Gierer and Meinhardt, 1972). It was also Gierer and Meinhardt that borrowed the term “lateral inhibition” from neuroscience, to refer to morphogenetic pattern formation.

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Lateral inhibition, as the name implies, is now taken to mean a process by which cells instruct adjacent cells to adopt a different fate than the instructing cell. The Notch pathway is a wellcharacterized mediator of lateral inhibition, yet it can also mediate lateral induction in which an instructing cell induces cells to adopt the same fate as the instructing cell. In this review, we discuss mechanisms controlling these precise lateral cues, focusing on organs in which these occur, and mechanisms that determine whether cues are inhibitory or inductive. NOTCH SIGNALING MECHANISMS Overview of Notch signaling The Notch signaling pathway is considered a relatively simple signaling system between two cells (Fig 1). The Notch receptor presented at the cell membrane of one cell binds a ligand presented at the surface of another contacting cell. Following receptor-ligand interaction the receptor is proteolytically processed at two distinct sites, resulting in liberation of the Notch intracellular domain (NICD), the active signaling fragment of the Notch pathway. Activation of one Notch receptor generates one signaling molecule, without further signal amplification. NICD translocates to the nucleus where it interacts with the transcriptional regulator CSL (“CBF-1, Suppressor of Hairless, LAG-2,” named after the mammalian, Drosophila, and C. elegans orthologues, also called RBP-Jκ in mammals). The interaction between NICD and CSL is followed by the assembly of co-activators to induce transcription, and the pathway itself is in fact modulated by a number of interacting factors (Andersson et al., 2011; Bray, 2016).

Notch receptors and ligands are single pass transmembrane proteins. Mammals express four different Notch receptor paralogs (Notch1-4) and five Notch ligands, belonging to the DSL (Delta, Serrate, Lag2) family (Mašek and Andersson, 2017). The ligands are divided into two categories 3

based on their structural resemblance to two ligands in Drosophila; Delta and Serrate, corresponding in mammals to Delta-like (Dll1, Dll3 and Dll4) or Jagged (Jagged1/Jag1 and Jagged2/Jag2), respectively. The strength of the Notch signal differs between receptors and is dependent on the activating ligand and also on the cellular and developmental context (GamaNorton et al., 2015; Van de Walle et al., 2013). Also, the redundancy between different ligands is cell context dependent (Preuße et al., 2015).

Notch receptors consist of 29-36 epidermal growth factor-like (EGF)-like domains, including the ligand interacting EGF 11-12 module, followed by three Lin-12-Notch repeats (LNR) and a hydrophobic region. Together these segments form the extracellular domain of the Notch receptor (NECD). The intracellular domain of the Notch receptor (NICD) consists of an RBP-Jκ associated molecule (RAM) domain, ankyrin repeats (ANK), two nuclear localization signal peptides (NLS) and a transactivation domain (TAD) including a proline (P)-glutamic acid (E) – serine (S)-threonine (T) (PEST) sequence. In addition, the transmembrane section contains the S3 cleavage site that is processed by γ-secretase following ligand binding, followed by shedding of the NECD. The LNR and hydrophobic region, which mediate heterodimerization, are positioned on the NECD and have been suggested to cover the S2 cleavage site, thereby preventing ligand-independent activation of the receptor (Sanchez-Irizarry et al., 2004). The intracellular RAM and ANK domains bind CSL and assemble transcription factors mediating Notch driven gene expression (Tamura et al., 1995), while the PEST domain is required for proteolytic degradation of NICD (Fryer et al., 2004; Öberg et al., 2001). Structurally Notch1 and Notch2 share many features, whereas Notch3 and Notch 4 differ in structure. Notch3 and Notch4 consist of fewer EGF-repeats and lack a conserved intracellular TAD domain (Kurooka et al., 1998; Lardelli et al., 1994; Uyttendaele et al., 1996).

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Like the receptors, Notch ligands are also single pass-transmembrane proteins containing several EGF-like domains. N-terminally, the DSL-domain and the two first EGF-like repeats are essential for receptor-ligand interaction (Parks et al., 2006; Shimizu et al., 1999). These EGF-like repeats are referred to as the Delta and OSM-11 (DOS) domain (Komatsu et al., 2008). Deltalike and Jagged ligands differ in that Jagged ligands contain more EGF-like repeats and also have a cysteine-rich domain close to the transmembrane domain. On the intracellular side the ligands comprise a C-terminal PDZ (PSD-95/Dlg/ZO-1) binding domain, which functions as an interaction module with other intracellular proteins. However, Jagged2 and Dll3 lack this feature. The amino acid structures of the PDZ motifs differ between Jagged and Dll and therefore they have different interaction partners (Adam et al., 2013). Thus there are fundamental differences in structure between Serrate and Delta type ligands.

The fact that Notch signaling is carried out by membrane-bound receptors and ligands makes this pathway ideally suited to establish patterning boundaries. Notch signal activation can either repress the expression of Notch ligands in signal-receiving cells (Fig 1B) resulting in lateral inhibition (Fig 1C), or induce the expression of Notch ligands in signal-receiving cells (Fig 1D), resulting in lateral induction (Fig1 E). This straight-forward model explains a number of patterning events across different model organisms, yet there are also caveats and exceptions to these models which will be described in further detail below.

FINE-TUNING THE NOTCH SIGNAL TO MODIFY LATERAL CUES

Notch signaling is at first glance a straight-forward signaling mechanism, dictated by a receptor interacting with a ligand. Yet a plethora of modifying mechanisms act on the Notch pathway to

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dictate signaling outcomes, including whether the ligand is presented in trans (by a contacting cell), or in cis (by the same cell), and whether specific enzymes modify receptor and/or ligand affinities. In addition, signaling range is suggested to be extended by filiopodia presenting ligands at a distance, while signaling intensity is dictated by contacting cell surface area. Combining these different regulatory mechanisms, a wealth of patterning mechanisms can arise from a deceptively simple receptor/ligand interaction.

Presentation mode dictates outcome: Trans and cis interactions Notch receptors interact with ligands presented by contacting cells as well as ligands within the same cell. Receptor-ligand interaction between two distinct cells is called trans-interaction, whereas cell autonomous interactions are called cis-interactions. The general view is that cisinteractions have an inhibitory function and counteract trans-activation of Notch signaling (Becam et al., 2010; del Álamo et al., 2011; Jacobsen et al., 1998; Klein et al., 1997). During lateral inhibition, activation of Notch represses expression of Delta ligands in the activated cell, thereby inhibiting reciprocal Notch activation in contacting cells in order to create an organized speckled pattern. Cis-interactions are thought further define the sender versus receiver fate between cells. This is achieved through mutual inhibition of receptors and ligands presented in the same cell leading to potentiation of the signaling status of distinct cells (Sprinzak et al., 2010). Trans-interactions are also necessary for boundary formation through lateral induction. During lateral induction active Notch signaling exerts a positive feedback loop by enhancing the expression of Notch ligands (Ross and Kadesch, 2004), creating a cascade of Notch activation from one cell to the next.

The specific effects of cis-interaction between distinct receptor-ligand pairs are still unknown. To date Notch1 is mainly used to model the specific outcomes of cis- and trans-interactions (del 6

Álamo et al., 2011; Kakuda and Haltiwanger, 2017; LeBon et al., 2014; Sprinzak et al., 2010), however these models should not be used as generalizing principles for all Notch receptor or ligands. The cis-interactive capacity varies between different ligands and in vitro studies have even shown differences in cis-inhibitory potency between ligands belonging to the same ligandfamily. The cis-inhibitory potential of Dll4 is stronger in comparison to Dll1 (Preuße et al., 2015), possibly due to differences in affinity to the Notch1 receptor (Andrawes et al., 2013), which also may explain the non-redundant function of these two ligands during axial segmentation (Preuße et al., 2015). Cell autonomous interactions between Notch receptors and ligands have been reported to occur both at the cell surface and intracellularly. Dll3 functions as a non-productive ligand and does not activate Notch in trans, but efficiently cis-inhibits Notch signaling by sequestering Notch receptors and reducing activation in trans by other ligands (Ladi et al., 2005). Dll3 exerts its function in the Golgi apparatus, preventing exocytosis of Notch and targeting it for degradation (Chapman et al., 2011). Some studies indicate possible cell intrinsic activatory effects of ligands on Notch signaling, i.e. cis-activation. Cis-activation can be difficult to separate from trans-activation, but expression of Jag1 in Notch1 reporter cells enhances Notch activation in a co-culture assay with Dll4 expressing cells (Benedito et al., 2009). In addition, following Drosophila SOP division and asymmetric endosomal distribution of Notch and Delta into the pIIa cell, ligand dependent Notch activation has been reported to take place in Sara endosomes (Coumailleau et al., 2009). Similar cell intrinsic properties have observed in rat pulmonary artery smooth muscle cells, where Notch3 activation relies on intracellular interaction with Jag1 (Ghosh et al., 2011). Cell autonomous ligand activation of Notch is also proposed to play a role in T-cell proliferation (Guy et al., 2013).

In many cellular and developmental contexts Notch activation by different ligands functions as a binary switch between two cell fates. How this is achieved since both ligands gives rise to the same signaling fragment (Gama-Norton et al., 2015; Ong et al., 2008) is still unclear. Signal 7

intensity, in terms of the number of active NICD fragments, is a factor that can direct cells towards a certain fate (Gama-Norton et al., 2015). Jagged ligands are sometimes described as inhibitory or antagonistic in regard to Dll-mediated activation of Notch signaling. Part of this inhibitory potential can be accredited to cis-inhibition of Notch by Jagged, whereas competition and induction of a weaker NICD signal by Jagged can be regarded as inhibitory in terms of DllNotch. However, Jagged modification by Lfng and Mfng, which reduces Jagged mediated Notch signaling, is shown to reduce cis-interactions without altering Jagged-Notch binding affinity in trans. This implies the possibility of a trans-inhibitory function of Jagged.

Post-translational modifications fine-tune receptor/ligand affinity Notch receptors are post-translationally modified through phosphorylation, ubiquitylation, glycosylation and acetylation, to name a few (Brückner et al., 2000; Espinosa et al., 2003; Foltz et al., 2002; Fryer et al., 2004; Guarani et al., 2011; Moloney et al., 2000; Sjöqvist et al., 2014; Öberg et al., 2001). In this review we focus on post-translational modifications that affect receptor-ligand binding and affinity, thereby altering signaling strength. Modulation of receptor responsiveness is of special importance in cell populations where different ligands promote distinct cell fates.

The ability of Fringes to modulate ligand discrimination may be especially important during patterning and formation of boundaries in tissues expressing both Dll and Jagged ligands. Fringes have been implicated in specification of dorsal-ventral borders in e.g. Drosophila wings and vertebrate limbs. Fringe expression functions in positioning Notch-responsive cells in specific locations important for proper development (Cohen et al., 1997; Irvine et al., 1999; Laufer et al., 1997; Rodriguez-Esteban et al., 1997; Zhang and Gridley, 1998). In addition, Fringes have been implicated in angiogenesis (Benedito et al., 2009). The interchangeable state

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between tip and stalk cell is modulated by Fringes (Jakobsson et al., 2010; Venkatraman et al., 2016).

The Fringe family of proteins are β3-N-acetylglucosaminyltransferases exerting their function by coupling GlcNAc to O-fucose residues on EGF repeats of the Notch receptors. This modification of the receptors takes place in the Golgi apparatus. Mammals express three different Fringe paralogs; Lunatic Fringe (Lfng), Manic Fringe (Mfng) and Radical Fringe (Rfng). Targeted mutation or deletion of Lfng in mice results in disturbed somitogenesis and rostral-caudal patterning defects. The somites are aberrant in size and shape with possible bifurcation (Evrard et al., 1998; Zhang and Gridley, 1998) and appear similar to the phenotypes observed in mice lacking Dll1 and Dll3 (de Angelis et al., 1997; Kusumi et al., 1998) as expected from reduced Dllmediated signaling. Mfng or Rfng, however, are not required for embryonic development (Moran et al., 2009, 1999). Addition of GlcNAc to Notch receptors by Fringes alters the responsiveness of the receptor towards different ligands. The general view, with regards to Notch1, is that Fringe modification enhances Notch activation by Dll ligands but weakens activation of Notch by Jagged. However, this is not true for all Fringe paralogs, since modification of Notch1 by Rfng enhances signaling induced by both Dll1 and Jag1. Responsiveness of distinct receptors towards one specific type of ligand may be of importance for the establishment of distinct boundaries during different developmental processes (Barrantes et al., 1999; Fleming et al., 1997; Kim et al., 1995). The effects of Fringe modifications on Notch1 and Notch2 in response to different ligands are summarized in Table 1. Kakuda et al. (2017) (Kakuda and Haltiwanger, 2017) showed that all Fringes have distinct glycosylation patterns of Notch1, which either potentiate or reduce the Notch response in a ligand specific manner. EGF12 appears to be the most important site for GlcNAc addition by Lfng and Rfng, enhancing activation of Notch1 through both Dll1 and Jag1. Modification of other O-fucose residues by Lfng counteracts Jagged-induced Notch1 activation, and Lfng thereby exerts an overall negative effect on Jagged9

induced signaling. Dll4 has a high affinity for Notch1 and glycosylation of EGF12 does not enhance this interaction (Taylor et al., 2014). Glycosylation of Notch1 by Mfng functions solely by promoting Dll-signaling at the expense of Jagged (Benedito et al., 2009; Kakuda and Haltiwanger, 2017; LeBon et al., 2014). Mutations of specific O-fucosylations sites on Notch1 impacts the interaction between Dll1 and Notch1, whereas Jag1 binding to Notch1 is hardly affected (Kakuda and Haltiwanger, 2017). This suggests Fringes alter Jagged-mediated Notch activation through other mechanisms, subsequent to receptor interaction. One possible explanation is that Mfng or Lfng-mediated glycosylation weakens the interaction between Notch1 and Jag1, resulting in an unstable bond which cannot withstand the force required for revealing the S2 site for proteolytic processing by ADAM-metalloproteases. Furthermore, the potency of both Dll1 and Jag1 engagement in cis-interactions is modulated by Lfng and Mfng. Glycosylation of Notch1 exerts a similar response on signaling in cis as observed in trans, enhancing cisinhibition by Dll1 but weakening cis-inhibition by Jag1 (LeBon et al., 2014).

Notch2 is also a substrate for Fringe glycosylation. Modification by Lfng and Mfng enhance Dll1 activation of Notch2, but the effects on Jagged vary. Mfng has an inhibitory function (Benedito et al., 2009), but Lfng has been reported to both enhance and reduce Jag1 mediated activation of Notch2 (Hicks et al., 2000; Shimizu et al., 2001). EGF12 is a potential O-fucosylation site in all Notch receptors (Haines and Irvine, 2003) and thereby also constitutes a possible Fringe modification site on Notch2. Structurally Notch2 resembles Notch1, with 36 EGF repeats, but the chemical and mechanical requirements of Notch2 activation differs from those of Notch1 (Habets et al., 2015). Furin processing in the Golgi, differently affects Notch1 and Notch2 membrane expression and signaling (Gordon et al., 2009). In addition, there are indications of Notch2 being more susceptible to ligand mediated activation in comparison to Notch1 (Liu et al., 2013) possibly due to differences in unfolding of the NRR domain in order to reveal the S2 site to metalloproteases (Stephenson and Avis, 2012). It would be interesting to compare the forces 10

produced between different receptor-ligand pairs using magnetic tweezer systems (Gordon et al., 2015; Laurence et al., 2012) including Fringe modifications of the receptors.

Fringe modification of ligands

In addition to modifying Notch receptors, Fringe proteins also mediate GlcNAc addition to Notch ligands. The functional relevance of ligand modification by Fringes is still debated and at first glance the effects seemed very mild (Müller et al., 2014). However, expression of Lfng or Mfng in signal-sending cells reduces activation of Notch1 in cocultured cells (Benedito et al., 2009; Okubo et al., 2012). Cis-interactions between Notch1 and Dll1 are enhanced by Lfng (LeBon et al., 2014), reducing the amount of receptors and ligands available for signaling in trans. The Lfng-induced reduction of Dll1-mediated Notch signaling was further repressed by co-expression of Notch1 and Dll3 in the signal-sending cells (Okubo et al 2012) corroborating the cis-inhibitory function of Dll in response to Lfng. Furthermore, the cis-inhibitory function of Dll3 has been shown to be dependent on Fringe modification (Serth et al., 2015).

Increasing your impact – cell surface contact area and filopodia define the dynamic Notch patterning range

Notch ligands are typically studied in their capacity as membrane-bound proteins. In this context, the expected range of inhibitory signaling is limited to contacting cells, which in the simplest form allows patterning of one cell, and its surrounding immediately contacting cells, with a maximal pattern wavelength of 2 cells (Collier et al., 1996). Yet several developmental patterns regulated by Notch signaling do not follow this simple binary pattern. Other mechanisms must thus exist to fine-tune the level of signal-sending by ligand-expressing cells, and increasing the effective range of signaling. 11

If ligands are membrane-bound, signal strength can be determined by ligand presentation at the membrane; for example ligand density, clustering, presentation mode, or surface area of membrane presented. Indeed, mathematical modeling, in vitro assays and Drosophila midgut analyses confirm that cell surface contact area regulates Notch signaling strength (Guisoni et al., 2017; Shaya et al., 2017). Thus cell size determines cell fate decisions during lateral inhibition, wherein smaller cells are more likely to become signal-sending ligand-expressing cells. This also offers the possibility that Notch ligands could instruct different cell fates in contacting cells based on cell surface and cell-cell contact area. Thereby cells with large contacting areas would receive high Notch signaling and cells with small contact areas would receive low Notch signaling.

At least two modes of ligand presentation introduce wider signaling ranges: ligand secretion on exosomes or in soluble form, and long-range presentation of ligand on filopodia. Evidence for both has been described, with different effects on signaling. Soluble ligands are mainly thought to inhibit Notch signaling, though some examples of activating soluble ligands have been described. For example, in vascular development, Dll4 is packaged in endosomes that are delivered to other endothelial cells, where Dll4 cis-inhibits Notch activity (Sheldon et al., 2010). In contrast, an alternative transcript for Jag1, isolated from keratinocytes and encoding only the extracellular domain, was capable of inducing keratinocyte differentiation, suggesting long-range activation of Notch signaling by the soluble ligand (Aho, 2004). Long-range activation of Notch signaling can also be accomplished by filipodia (Cohen et al., 2010; de Joussineau et al., 2003), extending the range at which Notch ligands can accomplish lateral inhibition. Importantly, mathematical modeling shows that the increased signaling range permitted by filopodia allows a wide plethora of patterns to emerge simply from lateral inhibition mechanisms, including speckles, stripes, labyrinths and more (Hadjivasiliou et al., 2016). 12

Intriguingly, the fact that Notch ligands can act as short range activating signals when membrane-bound, but as inhibitors when secreted, introduces the possibility that Notch ligands could act as combined Turing morphogens with both activating and inhibiting capacities mediated by the same Notch ligand, depending on presentation and distribution mode.

LATERAL PATTERNING IN VIVO

Notch signaling, as a biological program to induce precise lateral cues, has been used reiteratively across different organisms, from flies to vertebrates (Table 2), to control development of a variety processes. Beginning from an initially equivalent cell population, cells that stochastically express slightly higher levels of ligand can induce different types of cell patterns depending on whether the signal-receiving cells interpret the signal with positive or negative feedback. In the case of lateral inhibition, minor fluctuations in receptor and ligand concentration are amplified by negative feedback when a ligand-expressing cell instructs contacting cells to downregulate Notch ligand. Conversely, in the case of lateral induction, positive feedback in the signal-receiving cells results in upregulation of Notch ligand, which then instructs yet other cells to also upregulated Notch ligand and adopt the same fate (Fig 1b-d).

The downstream circuitry mediating positive and negative feedback is well established for several model systems, where downregulation of Delta ligand is accomplished indirectly through induction of Hes/Hey Notch target genes that repress ligand-inducing neurogenic genes (Figure 2A), while the up-regulation of Jagged/Serrate ligands is direct (Figure 2B). Further information on this feedback is detailed in the individual examples listed below. Here, we describe three well-

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established models of lateral inhibition or lateral induction from Drosophila, C. elegans and mouse, though far more examples exist (Table 2).

Vulval/uterine development in C elegans.

C elegans, a roundworm whose bodyplan as an adult consists of 959 or 1,031 cells, depending on its sex, was championed by Sidney Brenner and John Sulston as an animal model in the 1960’s (Ankeny, 2001; Brenner, 1974; Nigon and Félix, 2017; Sulston and Brenner, 1974), for which they were awarded the Nobel Prize in 2002 together with H. Robert Horvitz (Nobelprize.org., 2014). It is an ideal model in which to study patterning, morphogenesis, and differentiation, as it is transparent and contains several organs similar to mammals, and the lineage of all cells has been exhaustively mapped (Sternberg, 2017; Sulston and Horvitz, 1977). Importantly, Notch signaling regulates a number of developmental steps in C. elegans, with both lateral induction and lateral inhibition activities (Priess, 2005).

The Notch repertoire in C. elegans shows some important differences with vertebrate Notch counterparts. C. elegans has two Notch receptors, LIN-12 and GLP-1, which can functionally replace one another during development (Struhl et al., 1993), and ten DSL/EGF Notch ligands: APX-1, ARG-1, DSL1-7, and LAG-2 (Greenwald, 2005; Kopan and Ilagan, 2009a; Mašek and Andersson, 2017). The best characterized of the ligands are LAG-2, APX-1 and DSL1. Importantly, LAG-2 and APX-1 are membrane-bound, while DSL-1 lacks a transmembrane domain and is thought to be secreted (Kopan and Ilagan, 2009b). In addition to the DSL/EGF ligands, there are DOS-containing co-ligands; DOS1-3, OSM-7 and OSM-11 (Kopan and Ilagan, 2009a). Another important difference is thus the requirement for membrane anchoring and endocytosis: while endocytosis is generally required in ligand-expressing cells to activate Notch signaling in for example Drosophila (Parks et al., 2000), its role in C. elegans seems less strong 14

(Komatsu et al., 2008), and secreted C. elegans Notch ligands are Notch-activation competent (Chen and Greenwald, 2004).

The majority of C elegans individuals are hermaphroditic, and vulval/uterine development can thus easily be observed, integrating precise proliferation, induction, lateral inhibition, differentiation, and morphogenesis cues to form the mature sexual organ (Schindler and Sherwood, 2013; Sternberg, 2005). Notch signaling controls several stages of vulval/uterine development (Fig 3). The master regulator of uterine and vulval development is the anchor cell (AC), which organizes their development and physically connects the epidermis to the uterus. The AC arises from lateral inhibition between two initially equivalent cells (a cells), in which lateral inhibition, mediated by LIN-12 and LAG-2, amplifies stochastic differences in ligand and receptor expression such that one cell, expressing higher LIN-12/Notch is inhibited from adopting the anchor cell fate, becoming instead a ventral uterine (VU) precursor cell ((Wilkinson et al., 1994) and Fig 3A). lin-12 and lag-1 (the CSL orthologue) are direct LIN-12/Notch target genes, and are upregulated in the VU cell. However, loss of lin-12/Notch in the AC also results in persistent lag-1 expression in the anchor cell, resulting in vulval-uterine connection defects. Therefore down-regulation of lag-1 in the anchor cell is also a crucial step in its differentiation (Park et al., 2013).

The vulva develops from three precursor cells that divide and differentiate into 22 cells of 7 celltypes. These cells then undergo complex morphogenetic movements, including invagination and lumenogenesis, to generate the vulva. The three original specialized cells are selected from a set of vulval precursor cells (VPCs), whose default differentiation state is to become a non-vulval epidermal cell (termed the 3° VPC fate). The alternative 1° VPC fate or 2° VPC fate is adopted by one or two cells, respectively, in response to a LIN-3/EGF signal secreted by the anchor cell positioned above the future 1° VPC. Ablation of the anchor cell using laser microsurgery in the 15

80’s showed that this cell is required for vulval induction (Kimble, 1981), specifically for induction of the 1° fate VPC. The 2° VPC fate is also dependent on Notch-mediated lateral inhibition of the 1° fate and lateral induction of the 2° fate, via DSL expressed on the 1° fate VPC ((Greenwald et al., 1983; Sternberg, 1988; Sternberg and Horvitz, 1989) and Fig 3B).

In addition to patterning the underlying vulval precursor cells, the LAG-2 expressing anchor cell inhibits adjacent LIN-12-expressing uterine cells from adopting the default rho state, which then instead adopt the π (pi) fate ((Newman et al., 1995) and Fig 3C). The π cells divide once, and eight cells contribute to the uterine seam syncytium, which attaches the uterus to lateral epithelial cells, while the remaining four stabilize the uterine-vulval connection by attaching to underlying vulval cells (Schindler and Sherwood, 2013).

How is such precise patterning maintained in spite of stochastic differences in cell positioning or signaling strength? Spatial variability could result in differences in signaling and anatomical variability. This is reduced by intercellular signaling pathways that position cells more precisely to improve cue interpretation (Grimbert et al., 2016; Huelsz-Prince and van Zon, 2017)

In vertebrates and in Drosophila, Fringe modification of Notch receptors biases Notch signaling. While genetic modifiers of Notch phenotypes have been identified (Greenwald and Kovall, 2013; Lehner et al., 2006), C. elegans appears to lack traditional Fringe homologs, but do harbor two divergent Fringe homologs (Yuan et al., 1997), which are not yet linked to Notch activity in C. elegans. In fact, Notch receptors and ligands in C. elegans appear to lack the majority of the required linkage motifs (Blair, 2000).

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Interestingly, a preprint manuscript describes characterization of the entire C. elegans L2 larva, using single-cell RNA sequencing (Cao et al., 2017). At this stage, the vulval precursor cells have already been born, but remain in an extended G1 phase until L3. Thus, the RNA sequencing, which identified VPCs with lin-12 and osm-11 expression, may also reveal previously unknown intrinsic differences, further adding to our understanding of the development of this organ. Coupling these studies with cell-specific proteome analyses could further enrich our understanding of how specific Notch components interact to control development in C elegans (Yuet et al., 2015).

In sum, Notch signaling mediates a series of lateral inhibition steps during vulval and uterine development, to select an anchor cell (repressing ventral uterine cell fate), to ensure that only one ventral precursor cell adopts the 1° VPC fate (by lateral inhibition of this fate in adjacent cells which instead adopt the 2° VPC fate), and by inducing a π fate in uterine cells which would otherwise have become rho cells.

Lateral inhibition and lateral induction in Drosophila SOPs

Much of what we know of lateral inhibition and lateral induction comes from Drosophila. An excellent model of Notch control of development via lateral cues is offered by sensory organ precursor development into macrochaetae. These mechanosensory bristles cover the adult Drosophila body and head in a strictly defined number and pattern, and are composed of 4 cells: a hair cell, a socket cell, a sheath cell and a neuron, that arise in a tightly regulated manner, from a single precursor cell termed the sensory organ precursor (SOP). SOPs are selected within proneural stripes through a lateral patterning mechanism mediated by Delta (Corson et al., 2017; Troost et al., 2015), though Serrate acts redundantly with Delta to control SOP selection (Pitsouli and Delidakis, 2005). Previously, lateral inhibition mechanisms were thought to be the 17

result of slightly higher levels of Delta on one cell inducing down-regulation of Delta expression in contacting cells. However, uniform re-expression of Delta in proneural clusters lacking Delta and Serrate rescues SOP selection, suggesting transcriptional down-regulation of Delta is not required for lateral inhibition (Pitsouli and Delidakis, 2005). In line with this, normal neuroblast segregation upon constitutive expression of Notch and Delta showed that feedback loops are not required for this process either (Seugnet et al., 1997). Instead, it has now been suggested that it is the activity of Delta, rather than its expression, that is regulated by lateral inhibition (Chanet et al., 2009). The subsequent SOP asymmetric cell divisions leading to binary cell fate choices are also mediated by Notch signaling, and have been recently reviewed elsewhere (Schweisguth, 2015).

Modification of Notch signaling through filopodia-mediated ligand presentation or Fringe modification of receptors or ligands could impact on SOP development. Although filopodia have been described to increase the range of Notch ligand presentation in Drosophila (Cohen et al., 2010; de Joussineau et al., 2003) and in zebrafish (Hamada et al., 2013), more recent studies suggest the inhibitory signal mediated by Delta extends only to the adjacent cell during SOP selection (Troost et al., 2015). Fringe regulatory function has largely been determined using overexpression studies in cells. To our knowledge, no role of Fringe function in SOP development has been described. However, Fringe-regulated lateral signaling is crucial for Drosophila wing and eye development, and chick spinal cord, as well as development of the mammalian inner ear (described in the next section).

During development of the Drosophila wing, active Notch signaling is constrained to the border between a dorsal cell compartment expressing Fringe and Serrate and a ventral compartment expressing Delta. The presence of Fringe in Serrate-positive cells reduces Notch signaling between these cells whereas lack of Fringe functions similarly in cells expressing Delta, leading 18

to high Notch activity at the boundary between the dorsal and ventral sections (Irvine and Wieschaus, 1994; Wu and Rao, 1999). Similar segregation of Fringe together with Serrate and not with Delta is observed in the developing eye of Drosophila, which relies on a dorsoventral boundary of active Notch signaling in order to develop normally (Choi and Cho, 1998; Domínguez and de Celis, 1998). In chick spinal cord however, Fringe localizes with Dll1, leading to reduced signaling across the dorsoventral axis and restricting Notch signaling to specific progenitor domains (Marklund et al., 2010). In sum, Fringe control of lateral signaling processes is highly dependent on whether it is expressed in ligand or receptor-expressing cells.

Inner ear patterning by lateral induction and lateral inhibition

Development of the auditory sensory organ, the organ of Corti, and cell fate specification into mechanosensing hair cells versus non-sensory supporting cells is regulated by Notch signaling (Fig 4 and (Martin L. Basch et al., 2016; Kiernan, 2013)). The organ of Corti consists of a rigorously patterned set of cells that mediate sensory capacity, structural support and amplification; with a single row of inner hair cells and three rows of outer hair cells extending along the cochlear duct, surrounded by different supporting cells, the development of which is an excellent model of vertebrate lateral patterning.

Notch1 was the first receptor identified to be highly expressed during inner ear development (Lanford et al., 1999; Lewis et al., 1998), though more recently it has become clear that supporting cells also express Notch2 and Notch3 (Maass et al., 2015; Rg Waldhaus et al., 2015; Scheffer et al., 2015).

Notch signaling drives the development of the organ of Corti through lateral induction, and subsequent rounds of lateral inhibition. First, around embryonic day (E) 11.5 in mouse, Jag1 19

expression is initiated in the epithelial cells of the otic vesicle leading to the establishment of prosensory patches through lateral induction (Fig 4, panel1, (Kiernan et al., 2006, 2001; Morrison et al., 1999)). Next, Jag1 and Jag2 define the boundary of the organ of Corti through lateral inhibition, repressing an organ of Corti fate in Kölliker’s organ (Fig 4, panel 2, (Martin L Basch et al., 2016)), adjacent to the inner hair cell row. In a final round of lateral inhibition, Jag2 and Dll1 in post-mitotic prosensory cells repress the primary hair cell fate in adjacent cells, which instead develop into supporting cells (Fig 4, panel 3, (Kiernan et al., 2005; Lanford et al., 1999)). Thus, the final structure of the organ of Corti (Fig 4, panel 4) arises after a sequence of finely regulated Notch signaling events.

These patterning and differentiation steps are fine-tuned by different dose-dependencies of the individual events. For example, low levels of Jag1-driven Notch signaling compete with and antagonize stronger levels of Dll1-driven Notch signaling, thus reducing Notch signaling activity and promoting hair cell differentiation (Petrovic et al., 2014). Fringes also act to fine-tune several steps of inner ear development, with expression of Lfng and Mfng enriched in different cells in cochlear development. During boundary establishment, co-expression of Lfng and Mfng creates a margin of cells unresponsive to the Jag1-mediated signal supplied by surrounding cells, thus inhibiting adjacent prosensory cells of the Köllikers organ from adopting hair cell or phalangeal cell fates. Lfng is expressed with Jag1 prior to hair cell differentiation and later on in the supporting cells, maintaining low levels of Notch signaling (Martin L Basch et al., 2016; Burns et al., 2015; Zhang et al., 2000). Mfng, on the other hand, is specifically found in hair cells (Martin L Basch et al., 2016; Burns et al., 2015). However, in absence of Lfng and Mfng the balance of signal receiving vs. signal sending is perturbed resulting in an increase in hair cell differentiation (Martin L Basch et al., 2016; LeBon et al., 2014), similar to the phenotype observed in Jagged2 KO mice (Martin L Basch et al., 2016; Lanford et al., 1999). Based on this work, Basch and colleagues proposed the occurrence of a binary cell fate decision taking place after the initial 20

lateral inhibition, wherein the redistribution of Mfng and Lfng into different cells determines the cell fate choice of hair cell versus inner phalangeal cell (Martin L Basch et al., 2016). Finally, it is worth noting that Notch2, which is now known to be expressed in supporting cells (Maass et al., 2015; Rg Waldhaus et al., 2015; Scheffer et al., 2015), may respond differently to Fringe modifications compared to Notch1 (Hicks et al., 2000) and it is thus an open question whether the segregation of Lfng and Mfng to distinct cell populations impacts on signaling from other receptors than Notch1, to regulate differentiation in the inner ear.

While this three-step model for Notch control of cochlear development is supported by a wealth of data from loss of function and overexpression studies, some experiments contradict the model, or offer complementary explanations. Ear-specific deletion of the Notch effector RBPJk/CSL using Pax2-cre results in a shorter cochlea, but does not impact on prosensory patch establishment, which is thought to be mediated by Jag1-induced lateral induction (Basch et al., 2011). Deletion of RBPJ using Foxg1-cre resulted in a severe, but not complete, loss of prosensory patches, suggesting that the function of Notch signaling may be to maintain rather than initiate prosensory patch formation. However, although both Pax2-cre (Ohyama and Groves, 2004) and Foxg1-cre (Hébert and McConnell, 2000) are active from E8.5, it is unclear how long it takes to completely clear residual RBPJk mRNA and protein from cells in the otic vesicle, thus it is possible that a small remaining pool of RBPJk is sufficient to mediate prosensory patch induction.

In conclusion, the inner ear is a structure meticulously patterned by lateral induction and lateral inhibition cues, ideally suited to studying the effects of minor perturbations of Notch signaling strength and induction/inhibition capacity on boundary formation and cell fate determination in a mammalian model.

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CONCLUSION Lateral cues are essentials for mediating precise signals to pattern organisms with single cell resolution. The Notch signaling pathway, which comprises membrane-bound receptors and ligands (in most model organisms) is ideally suited to mediating cell-cell patterning mechanisms. While lateral inhibition and lateral induction either inhibit or induce Notch ligand expression in signal-receiving cells respectively, it is becoming less clear whether inhibition mechanisms solely repress a primary fate, or whether they also induce a secondary fate. For example, in the inner ear, Notch signaling, which represses the primary hair cell fate, has been suggested to also activate the supporting cell differentiation program (Campbell et al., 2016). It will be interesting to determine whether this effect is directly regulated by Notch signaling, for example whether relevant target genes contain CSL-binding sites, or whether this effect is downstream of inhibited hair cell fate. The lateral inhibition model has typically invoked indirect transcriptional down-regulation of Notch ligands as part of the mechanism of inhibition (Fig 2). Several lines of evidence now indicate that the inhibition of ligand activity in Notch signal-receiving cells may be independent of transcription. Constitutive expression of Notch and Delta do not inhibit normal neuroblast segregation (Seugnet et al., 1997) and persistent expression of Notch ligands does not abrogate lateral inhibition in SOP selection (Pitsouli and Delidakis, 2005), suggesting ligand expression down-regulation is not required for lateral inhibition. Mathematical modeling and in vitro experiments confirm that transcriptional down-regulation of Notch ligand is not required for lateral inhibition, rather Fringe expression likely increases trans-Notch activation by ligands, while also increasing cis-inhibition by ligand (LeBon et al., 2014; Matsuda et al., 2015). Thus Delta activity, rather than expression, is regulated by lateral inhibition (Chanet et al., 2009).

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What determines whether cues are interpreted with negative or positive feedback, resulting in lateral inhibition or lateral induction? Initially, the observation that many lateral induction processes involve Serrate/Jag1, while lateral inhibition processes involve Delta/Delta-like ligands, suggested that the two ligands induce different responses. Although both ligands activate Notch signaling, some important differences explain how opposite outcomes can be achieved. First of all, the Notch signaling pathway is particularly dose-sensitive, and different signal strengths can drive different outcomes (Gama-Norton et al., 2015). In general, Jag1 induces weaker Notch activation than Dll4 does (Gama-Norton et al., 2015; Petrovic et al., 2014), in spite of generally greater affinity of Notch receptors for Jag1. Thus low Jag1/Notch signaling can drive one response, while high Dll4/Notch signaling can drive a different response. The differential affinity further suggests a model in which high Notch signal strength, driven by Dll4, is in effect inhibited by Jag1 - reducing Notch signal strength to Jag1-driven signal strength (Gama-Norton et al., 2015; Petrovic et al., 2014). Second of all, Serrate/Jag1 is a direct Notch target gene (Manderfield et al., 2012), which amplifies Jag1/Notch signaling and can induce a wave of Jag1 expression and Notch activation ((Petrovic et al., 2014) and (Fig 1D,E)). Jag1 expression is thus under the control of Notch activation, with intron 2 of Jag1 containing a Notchresponsive RBPJ/CSL binding site (Manderfield et al., 2012). This control likely interacts with a number of other pathways to dictate whether Jag1 expression is induced in response to Notch activation, for example via p63-binding sites in intron 2 (Ross and Kadesch, 2004; Sasaki et al., 2002), upstream Wnt/b-catenin TCF/LEF binding sites (Estrach et al., 2006; Katoh and Katoh, 2006), and NFkb- or AP1-binding sites (Johnston et al., 2009) that activate Jag1-transcription in a cell-type dependent manner. These interacting pathways, and others yet to be discovered, may contribute to the likelihood that Jag1 is upregulated in response to Notch activation. Interestingly, the Jag1 ICD has also been suggested to activate transcription of AP-1 responsive elements (LaVoie and Selkoe, 2003), which may contribute to why Jag1 would be able to induce Jag1 in Notch/Jag1-expressing cells. This interaction remains to be tested. 23

Another avenue of exciting future research concerns the roles of the extracellular and intracellular domains of Notch ligands in determining signaling outcome. Previous studies swapping the ICDs of Notch receptors in vivo showed little effect on development, suggesting the ICDs of Notch1 and Notch2 are largely interchangeable (Liu et al., 2015). Similar studies, swapping for example the Jag1 ICD with the Jag2 ICD would be of interest to determine whether the Jag1-specific PDZ-binding domain contributes to dictating functional outcome, since Jag2 is instead associated with lateral inhibition (Kiernan et al., 2005; Lanford et al., 1999). However, other factors likely contribute to ligand-specific effects. Dll1 and Dll4 both contain a PDZ-binding domain, yet experiments in which Dll4 is knocked into the Dll1 locus showed that Dll4 is not sufficient to recapitulate Dll1 function (Preuße et al., 2015). Instead, Dll1-dependent somitogenesis is disrupted since Dll4 is a more efficient cis-inhibitor than Dll1 (Preuße et al., 2015). Swapping only the intra- or extra-cellular domains of Dll1/Dll4 would help resolve where this cis-inhibitory efficiency arises. Some exceptions and modifiers, to this generalization of Jag1/Serrate induction and Delta/inhibition function, exist. Serrate can act redundantly with Delta to mediate lateral inhibition (Pitsouli and Delidakis, 2005), indicating Serrate/Jag1 can mediate both lateral induction and lateral inhibition. Furthermore, the affinity of Notch receptors for Serrate/Jagged or Delta ligands is modulated by Fringes. Glycosylation of Notch receptor by Fringe during lateral inhibition generally enhances binding by Delta, increasing trans-activation and cis-inhibition by this ligand (LeBon et al., 2014; Matsuda et al., 2015). Simultaneously, glycosylation of Notch receptor reduces binding to Serrate/Jagged ligands, which would weaken lateral induction processes. However, Fringe modification of Notch signaling is more complex than this straight-forward generalization (Harvey et al., 2016; Kakuda and Haltiwanger, 2017), and it has been suggested that cells may adapt a hybrid state during Notch activation, as both signal-sending and signal-

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receiving, and that the outcome is dependent on Fringe modification and which ligand is dominant (Boareto et al., 2015). Another exciting aspect of Notch signaling, which is outside the scope of this review, is the role of signal oscillation in determining functional output. Notch oscillations are important in both the nervous system and in vertebral segmentation (Shimojo and Kageyama, 2016), where oscillation amplitude and duration impact on embryonic development. Thus, the output of the Notch pathway, in which Serrate/Jag1 is a direct Notch target, and Delta repression is an indirect downstream target, may be determined by pathway oscillation duration, or amplitude, to determine which portion of this circuitry is favored. There are thus several outstanding questions which would be of interest to resolve: –Is Serrate/Jag1 always initially up-regulated upon Notch activation? If not, what determines cellular competence to activate Jag1 transcription? Is Delta in fact always down-regulated during lateral inhibition, is this required, or is the protein activity inhibited, as has been suggested? Future studies to resolve these questions in a high throughput fashion will help guide our understanding of how lateral induction vs inhibition is determined. Patterning can be accomplished by numerous different types of molecules. Other than delimited cell fate choices such as SOP selection, or hair cell fate suppression, it is becoming clear that Notch signaling also induces larger patterns (Corson et al., 2017). For example, stripe formation in zebrafish has been suggested to follow a Turing pattern mechanism, with short range inhibition and long-range activation (Nakamasu et al., 2009), wherein the short range activation may be accomplished by Notch ligand on filopodia (Hamada et al., 2013). Originally, the reaction-diffusion model proposed a patterning mechanism whereby a rapidly diffusing inhibitor establishes a pattern together with a slowly diffusing activator (Turing, 1952). It is thus interesting to remember that secreted Notch ligands are thought to inhibit Notch signaling, while short-range membrane-bound or filopodia-bound ligand would activate Notch signaling. Thus, with a single gene, spliced to include or exclude the trans-membrane and intracellular domain, 25

nature could accomplish both long-range inhibition and short range induction. Adding in the finetuning by different Notch ligands, or Fringe modifications, a plethora of possible pattern formation mechanisms arise. Future efforts to elucidate how the entire Notch repertoire is integrated to mediate patterning will likely reveal new mechanisms determining Notch-mediated lateral inhibition and induction.

ACKNOWLEDGEMENTS/GRANT SUPPORT

ERA and members of Andersson lab were supported by a Center of Innovative Medicine (CIMED) Grant, the Daniel Alagille Award, KI Funding and Alex and Eva Wallström Foundation. MS was supported by an OSK Huttunen post doc fellowship. Figures by Mattias Karlen (http://mattiaskarlen.se/) based on drafts prepared by MS and ERA, and we are grateful to Peter Swoboda (KI) for feedback on the C. elegans figure and figure legend.

REFERENCES Abe, N., Hozumi, K., Hirano, K., Yagita, H., Habu, S., 2010. Notch ligands transduce different magnitudes of signaling critical for determination of T-cell fate. Eur. J. Immunol. 40, 2608– 17. doi:10.1002/eji.200940006 Adam, M.G., Berger, C., Feldner, A., Yang, W.-J., Wüstehube-Lausch, J., Herberich, S.E., Pinder, M., Gesierich, S., Hammes, H.-P., Augustin, H.G., Fischer, A., 2013. Synaptojanin-2 binding protein stabilizes the Notch ligands DLL1 and DLL4 and inhibits sprouting angiogenesis. Circ. Res. 113, 1206–18. doi:10.1161/CIRCRESAHA.113.301686 26

Aho, S., 2004. Soluble form of Jagged1: Unique product of epithelial keratinocytes and a regulator of keratinocyte differentiation. J. Cell. Biochem. 92, 1271–1281. doi:10.1002/jcb.20125 Andersson, E.R., Sandberg, R., Lendahl, U., 2011. Notch signaling: simplicity in design, versatility in function. Development 138, 3593–3612. doi:10.1242/dev.063610 Andrawes, M.B., Xu, X., Liu, H., Ficarro, S.B., Marto, J.A., Aster, J.C., Blacklow, S.C., 2013. Intrinsic selectivity of notch 1 for delta-like 4 over delta-like 1. J. Biol. Chem. 288, 25477– 25489. doi:10.1074/jbc.M113.454850 Ankeny, R.A., 2001. The natural history of Caenorhabditis elegans research. Nat. Rev. Genet. 2, 474–9. doi:10.1038/35076538 Barrantes, I.B., Elia, A.J., Wünsch, K., Hrabe de Angelis, M.H., Mak, T.W., Rossant, J., Conlon, R.A., Gossler, A., de la Pompa, J.L., 1999. Interaction between Notch signalling and Lunatic fringe during somite boundary formation in the mouse. Curr. Biol. 9, 470–80. Basch, M.L., Brown, R.M., Jen, H.-I., Semerci, F., Depreux, F., Edlund, R.K., Zhang, H., Norton, C.R., Gridley, T., Cole, S.E., Doetzlhofer, A., Maletic-Savatic, M., Segil, N., Groves, A.K., 2016. Fine-tuning of Notch signaling sets the boundary of the organ of Corti and establishes sensory cell fates. Elife 5, 841–850. doi:10.7554/eLife.19921 Basch, M.L., Brown, R.M., Jen, H.I., Groves, A.K., 2016. Where hearing starts: The development of the mammalian cochlea. J. Anat. 228, 233–254. doi:10.1111/joa.12314 Basch, M.L., Ohyama, T., Segil, N., Groves, A.K., 2011. Canonical Notch Signaling Is Not Necessary for Prosensory Induction in the Mouse Cochlea: Insights from a Conditional Mutant of RBPjκ. J. Neurosci. 31. Becam, I., Fiuza, U.-M., Arias, A.M., Milán, M., 2010. A role of receptor Notch in ligand cis-

27

inhibition in Drosophila. Curr. Biol. 20, 554–60. doi:10.1016/j.cub.2010.01.058 Benedito, R., Roca, C., Sörensen, I., Adams, S., Gossler, A., Fruttiger, M., Adams, R.H., 2009. The notch ligands Dll4 and Jagged1 have opposing effects on angiogenesis. Cell 137, 1124–35. doi:10.1016/j.cell.2009.03.025 Blair, S.S., 2000. Notch signaling: Fringe really is a glycosyltransferase. Curr. Biol. 10, R608– R612. doi:10.1016/S0960-9822(00)00633-3 Boareto, M., Jolly, M.K., Lu, M., Onuchic, J.N., Clementi, C., Ben-Jacob, E., 2015. Jagged–Delta asymmetry in Notch signaling can give rise to a Sender/Receiver hybrid phenotype. Proc. Natl. Acad. Sci. 112, E402–E409. doi:10.1073/pnas.1416287112 Bray, S.J., 2016. Notch signalling in context. Nat. Rev. Mol. Cell Biol. 9. doi:10.1038/nrm.2016.94 Brenner, S., 1974. The genetics of Caenorhabditis elegans. Genetics 77, 71–94. Brückner, K., Perez, L., Clausen, H., Cohen, S., 2000. Glycosyltransferase activity of Fringe modulates Notch-Delta interactions. Nature 406, 411–415. doi:10.1038/35019075 Burns, J.C., Kelly, M.C., Hoa, M., Morell, R.J., Kelley, M.W., 2015. Single-cell RNA-Seq resolves cellular complexity in sensory organs from the neonatal inner ear. Nat. Commun. 6, 8557. doi:10.1038/ncomms9557 Campbell, D.P., Chrysostomou, E., Doetzlhofer, A., 2016. Canonical Notch signaling plays an instructive role in auditory supporting cell development. Sci. Rep. 6, 19484. doi:10.1038/srep19484 Cao, J., Packer, J.S., Ramani, V., Cusanovich, D.A., Huynh, C., Daza, R., Qiu, X., Lee, C., Furlan, S.N., Steemers, F.J., Adey, A., Waterston, R.H., Trapnell, C., Shendure, J., 2017. Comprehensive single cell transcriptional profiling of a multicellular organism by 28

combinatorial indexing. bioRxiv. Chanet, S., Vodovar, N., Mayau, V., Schweisguth, F., 2009. Genome engineering-based analysis of Bearded family genes reveals both functional redundancy and a nonessential function in lateral inhibition in Drosophila. Genetics 182, 1101–8. doi:10.1534/genetics.109.105023 Chapman, G., Sparrow, D.B., Kremmer, E., Dunwoodie, S.L., 2011. Notch inhibition by the ligand Delta-Like 3 defines the mechanism of abnormal vertebral segmentation in spondylocostal dysostosis. Hum. Mol. Genet. 20, 905–916. doi:10.1093/hmg/ddq529 Chen, N., Greenwald, I., 2004. The lateral signal for LIN-12/Notch in C. elegans vulval development comprises redundant secreted and transmembrane DSL proteins. Dev. Cell 6, 183–92. doi:10.1016/S1534-5807(04)00021-8 Choi, K.-W., Cho, K.-O., 1998. Fringe is essential for mirror symmetry and morphogenesis in the Drosophila eye. Nature 396, 272–276. doi:10.1038/24394 Cohen, B., Bashirullah, A., Dagnino, L., Campbell, C., Fisher, W.W., Leow, C.C., Whiting, E., Ryan, D., Zinyk, D., Boulianne, G., Hui, C., Gallic, B., Phillips, R.A., Lipshitz, H.D., Egan, S.E., 1997. Fringe boundaries coincide with Notch-dependent patterning centres in mammals and alter Notch-dependent development in Drosophila. Nat. Genet. 16, 283–288. doi:10.1038/ng0797-283 Cohen, M., Georgiou, M., Stevenson, N.L., Miodownik, M., Baum, B., 2010. Dynamic Filopodia Transmit Intermittent Delta-Notch Signaling to Drive Pattern Refinement during Lateral Inhibition. Dev. Cell 19, 78–89. doi:10.1016/j.devcel.2010.06.006 Collier, J.R., Monk, N.A.M., Maini, P.K., Lewis, J.H., 1996. Pattern Formation by Lateral Inhibition with Feedback: a Mathematical Model of Delta-Notch Intercellular Signalling. J. Theor. Biol. 183, 429–446. doi:10.1006/jtbi.1996.0233 29

Corson, F., Couturier, L., Rouault, H., Mazouni, K., Schweisguth, F., 2017. Self-organized Notch dynamics generate stereotyped sensory organ patterns in Drosophila. Science (80-. ). 356, eaai7407. doi:10.1126/science.aai7407 Coumailleau, F., Fürthauer, M., Knoblich, J. a, González-Gaitán, M., 2009. Directional Delta and Notch trafficking in Sara endosomes during asymmetric cell division. Nature 458, 1051– 1055. doi:10.1038/nature07854 D’Amato, G., Luxán, G., Del Monte-Nieto, G., Martínez-Poveda, B., Torroja, C., Walter, W., Bochter, M.S., Benedito, R., Cole, S., Martinez, F., Hadjantonakis, A.-K., Uemura, A., Jiménez-Borreguero, L.J., de la Pompa, J.L., 2015. Sequential Notch activation regulates ventricular chamber development. Nat. Cell Biol. 18, 7–20. doi:10.1038/ncb3280 de Angelis, M.H., Mclntyre, J., Gossler, A., 1997. Maintenance of somite borders in mice requires the Delta homologue Dll1. Nature 386, 717–721. doi:10.1038/386717a0 de Joussineau, C., Soul?, J., Martin, M., Anguille, C., Montcourrier, P., Alexandre, D., 2003. Delta-promoted filopodia mediate long-range lateral inhibition in Drosophila. Nature 426, 555–559. doi:10.1038/nature02157 del Álamo, D., Rouault, H., Schweisguth, F., 2011. Mechanism and significance of cis-inhibition in Notch signalling. Curr. Biol. 21, R40-7. doi:10.1016/j.cub.2010.10.034 Domínguez, M., de Celis, J.F., 1998. A dorsal/ventral boundary established by Notch controls growth and polarity in the Drosophila eye. Nature 396, 276–8. doi:10.1038/24402 Espinosa, L., Inglés-Esteve, J., Aguilera, C., Bigas, A., 2003. Phosphorylation by glycogen synthase kinase-3β down-regulates Notch activity, a link for Notch and Wnt pathways. J. Biol. Chem. 278, 32227–32235. doi:10.1074/jbc.M304001200 Estrach, S., Ambler, C.A., Lo Celso, C. Lo, Hozumi, K., Watt, F.M., 2006. Jagged 1 is a β-

30

catenin target gene required for ectopic hair follicle formation in adult epidermis. Development 133. Evrard, Y.A., Lun, Y., Aulehla, A., Gan, L., Johnson, R.L., 1998. lunatic fringe is an essential mediator of somite segmentation and patterning. Nature 394, 377–381. doi:10.1038/28632 Fleming, R.J., Gu, Y., Hukriede, N.A., 1997. Serrate-mediated activation of Notch is specifically blocked by the product of the gene fringe in the dorsal compartment of the Drosophila wing imaginal disc. Development 124, 2973–81. Foltz, D.R., Santiago, M.C., Berechid, B.E., Nye, J.S., 2002. Glycogen synthase kinase-3beta modulates notch signaling and stability. Curr. Biol. 12, 1006–1011. doi:S0960982202008886 [pii] Fryer, C.J., White, J.B., Jones, K.A., 2004. Mastermind recruits CycC:CDK8 to phosphorylate the Notch ICD and coordinate activation with turnover. Mol. Cell 16, 509–520. doi:10.1016/j.molcel.2004.10.014 Gama-Norton, L., Ferrando, E., Ruiz-Herguido, C., Liu, Z., Guiu, J., Islam, A.B.M.M.K., Lee, S.U., Yan, M., Guidos, C.J., López-Bigas, N., Maeda, T., Espinosa, L., Kopan, R., Bigas, A., 2015. Notch signal strength controls cell fate in the haemogenic endothelium. Nat. Commun. 6, 8510. doi:10.1038/ncomms9510 Ghosh, S., Paez-Cortez, J.R., Boppidi, K., Vasconcelos, M., Roy, M., Cardoso, W., Ai, X., Fine, A., 2011. Activation dynamics and signaling properties of Notch3 receptor in the developing pulmonary artery. J. Biol. Chem. 286, 22678–87. doi:10.1074/jbc.M111.241224 Gierer, A., Meinhardt, H., 1972. A theory of biological pattern formation. Kybernetik 12, 30–9. Gordon, W.R., Vardar-Ulu, D., L’Heureux, S., Ashworth, T., Malecki, M.J., Sanchez-Irizarry, C., McArthur, D.G., Histen, G., Mitchell, J.L., Aster, J.C., Blacklow, S.C., 2009. Effects of S1

31

cleavage on the structure, surface export, and signaling activity of human Notch1 and Notch2. PLoS One 4, 12. doi:10.1371/journal.pone.0006613 Gordon, W.R.R., Zimmerman, B., He, L., Miles, L.J.J., Huang, J., Tiyanont, K., McArthur, D.G.G., Aster, J.C.C., Perrimon, N., Loparo, J.J.J., Blacklow, S.C.C., 2015. Mechanical Allostery: Evidence for a Force Requirement in the Proteolytic Activation of Notch. Dev. Cell 33, 729–736. doi:10.1016/j.devcel.2015.05.004 Greenwald, I., 2005. LIN-12/Notch signaling in C. elegans. WormBook 1–16. doi:10.1895/wormbook.1.10.1 Greenwald, I., Kovall, R., 2013. Notch signaling: genetics and structure. WormBook 1–28. doi:10.1895/wormbook.1.10.2 Greenwald, I.S., Sternberg, P.W., Robert Horvitz, H., 1983. The lin-12 locus specifies cell fates in caenorhabditis elegans. Cell 34, 435–444. doi:10.1016/0092-8674(83)90377-X Grimbert, S., Tietze, K., Barkoulas, M., Sternberg, P.W., Félix, M.-A., Braendle, C., 2016. Anchor cell signaling and vulval precursor cell positioning establish a reproducible spatial context during C. elegans vulval induction. Dev. Biol. 416, 123–135. doi:10.1016/j.ydbio.2016.05.036 Guarani, V., Deflorian, G., Franco, C.A., Krüger, M., Phng, L.-K., Bentley, K., Toussaint, L., Dequiedt, F., Mostoslavsky, R., Schmidt, M.H.H., Zimmermann, B., Brandes, R.P., Mione, M., Westphal, C.H., Braun, T., Zeiher, A.M., Gerhardt, H., Dimmeler, S., Potente, M., 2011. Acetylation-dependent regulation of endothelial Notch signalling by the SIRT1 deacetylase. Nature 473, 234–8. doi:10.1038/nature09917 Guisoni, N., Martinez-Corral, R., Garcia-Ojalvo, J., de Navascués, J., 2017. Diversity of fate outcomes in cell pairs under lateral inhibition. Development 144.

32

Guy, C.S., Vignali, K.M., Temirov, J., Bettini, M.L., Overacre, A.E., Smeltzer, M., Zhang, H., Huppa, J.B., Tsai, Y.-H., Lobry, C., Xie, J., Dempsey, P.J., Crawford, H.C., Aifantis, I., Davis, M.M., Vignali, D.A.A., 2013. Distinct TCR signaling pathways drive proliferation and cytokine production in T cells. Nat. Immunol. 14, 262–270. doi:10.1038/ni.2538 Habets, R.A.J., Groot, A.J., Yahyanejad, S., Tiyanont, K., Blacklow, S.C., Vooijs, M., 2015. Human NOTCH2 Is Resistant to Ligand-independent Activation by Metalloprotease Adam17. J. Biol. Chem. 290, 14705–16. doi:10.1074/jbc.M115.643676 Hadjivasiliou, Z., Hunter, G.L., Baum, B., 2016. A new mechanism for spatial pattern formation via lateral and protrusion-mediated lateral signalling. - PubMed - NCBI. J. R. Soc. Interface 13, 20160484. doi:10.1098/rsif.2016.0484 Haines, N., Irvine, K.D., 2003. Glycosylation regulates Notch signalling. Nat. Rev. Mol. Cell Biol. 4, 786–797. doi:10.1038/nrm1228 Hamada, H., Watanabe, M., Lau, H.E., Nishida, T., Hasegawa, T., Parichy, D.M., Kondo, S., 2013. Involvement of Delta/Notch signaling in zebrafish adult pigment stripe patterning. Development 141. Harvey, B.M., Rana, N.A., Moss, H., Leonardi, J., Jafar-Nejad, H., Haltiwanger, R.S., 2016. Mapping Sites of O-Glycosylation and Fringe Elongation on Drosophila Notch. J. Biol. Chem. 291, 16348–60. doi:10.1074/jbc.M116.732537 Hébert, J.M., McConnell, S.K., 2000. Targeting of cre to the Foxg1 (BF-1) Locus Mediates loxP Recombination in the Telencephalon and Other Developing Head Structures. Dev. Biol. 222, 296–306. doi:10.1006/dbio.2000.9732 Hicks, C., Johnston, S.H., diSibio, G., Collazo, A., Vogt, T.F., Weinmaster, G., 2000. Fringe differentially modulates Jagged1 and Delta1 signalling through Notch1 and Notch2. Nat. Cell Biol. 2, 515–20. doi:10.1038/35019553 33

Huelsz-Prince, G., van Zon, J.S., 2017. Canalization of C. elegans Vulva Induction against Anatomical Variability. Cell Syst. 4, 219–230.e6. doi:10.1016/j.cels.2017.01.009 Irvine, K.D., Rauskolb, C., Correia, T., 1999. Fringe-dependent separation of dorsal and ventral cells in the Drosophila wing. Nature 401, 476–480. doi:10.1038/46786 Irvine, K.D., Wieschaus, E., 1994. fringe, a boundary-specific signaling molecule, mediates interactions between dorsal and ventral cells during Drosophila wing development. Cell 79, 595–606. doi:10.1016/0092-8674(94)90545-2 Jacobsen, T.L., Brennan, K., Arias, A.M., Muskavitch, M.A., 1998. Cis-interactions between Delta and Notch modulate neurogenic signalling in Drosophila. Development 125, 4531–40. Jakobsson, L., Franco, C. a, Bentley, K., Collins, R.T., Ponsioen, B., Aspalter, I.M., Rosewell, I., Busse, M., Thurston, G., Medvinsky, A., Schulte-Merker, S., Gerhardt, H., 2010. Endothelial cells dynamically compete for the tip cell position during angiogenic sprouting. Nat. Cell Biol. 12, 943–953. doi:10.1038/ncb2103 Johnston, D.A., Dong, B., Hughes, C.C.W., 2009. TNF induction of jagged-1 in endothelial cells is NFkappaB-dependent. Gene 435, 36–44. doi:10.1016/j.gene.2009.01.003 Kakuda, S., Haltiwanger, R.S., 2017. Deciphering the Fringe-Mediated Notch Code: Identification of Activating and Inhibiting Sites Allowing Discrimination between Ligands. Dev. Cell 40, 193–201. doi:10.1016/j.devcel.2016.12.013 Karp, X., Greenwald, I., 2003. Post-transcriptional regulation of the E/Daughterless ortholog HLH-2, negative feedback, and birth order bias during the AC/VU decision in C. elegans. Genes Dev. 17, 3100–11. doi:10.1101/gad.1160803 Katoh, M., Katoh, M., 2006. Notch ligand, JAG1, is evolutionarily conserved target of canonical WNT signaling pathway in progenitor cells. Int. J. Mol. Med. 17, 681–685.

34

Kiernan, A.E., 2013. Notch signaling during cell fate determination in the inner ear. Semin. Cell Dev. Biol. 24, 470–479. doi:10.1016/j.semcdb.2013.04.002 Kiernan, A.E., Ahituv, N., Fuchs, H., Balling, R., Avraham, K.B., Steel, K.P., Hrabé de Angelis, M., 2001. The Notch ligand Jagged1 is required for inner ear sensory development. Proc. Natl. Acad. Sci. U. S. A. 98, 3873–8. doi:10.1073/pnas.071496998 Kiernan, A.E., Cordes, R., Kopan, R., Gossler, A., Gridley, T., 2005. The Notch ligands DLL1 and JAG2 act synergistically to regulate hair cell development in the mammalian inner ear. Development 132, 4353–62. doi:10.1242/dev.02002 Kiernan, A.E., Xu, J., Gridley, T., 2006. The Notch ligand JAG1 is required for sensory progenitor development in the mammalian inner ear. PLoS Genet. 2, e4. doi:10.1371/journal.pgen.0020004 Kim, J., Irvine, K.D., Carroll, S.B., 1995. Cell recognition, signal induction, and symmetrical gene activation at the dorsal-ventral boundary of the developing drosophila wing. Cell 82, 795– 802. doi:10.1016/0092-8674(95)90476-X Kimble, J., 1981. Alterations in cell lineage following laser ablation of cells in the somatic gonad of Caenorhabditis elegans. Dev. Biol. 87, 286–300. doi:10.1016/0012-1606(81)90152-4 Klein, T., Brennan, K., Arias, A.M., 1997. An Intrinsic Dominant Negative Activity of Serrate That Is Modulated during Wing Development inDrosophila. Dev. Biol. 189, 123–134. doi:10.1006/dbio.1997.8564 Komatsu, H., Chao, M.Y., Larkins-Ford, J., Corkins, M.E., Somers, G.A., Tucey, T., Dionne, H.M., White, J.Q., Wani, K., Boxem, M., Hart, A.C., 2008. OSM-11 Facilitates LIN-12 Notch Signaling during Caenorhabditis elegans Vulval Development. PLoS Biol. 6, e196. doi:10.1371/journal.pbio.0060196

35

Kopan, R., Ilagan, M.X.G., 2009a. The Canonical Notch Signaling Pathway: Unfolding the Activation Mechanism. Cell 137, 216–233. doi:10.1016/j.cell.2009.03.045 Kopan, R., Ilagan, M.X.G., 2009b. The Canonical Notch Signaling Pathway: Unfolding the Activation Mechanism. Cell 137, 216–233. doi:10.1016/j.cell.2009.03.045 Kurooka, H., Kuroda, K., Honjo, T., 1998. Roles of the ankyrin repeats and C-terminal region of the mouse Notch1 intracellular region. Nucleic Acids Res. 26, 5448–5455. doi:10.1093/nar/26.23.5448 Kusumi, K., Lander, E.S., Sun, E., Kerrebrock, A.W., Bronson, R.T., Chi, D.-C., Bulotsky, M., Spencer, J.B., Birren, B.W., Frankel, W.N., 1998. The mouse pudgy mutation disrupts Delta homologue Dll3and initiation of early somite boundaries. Nat. Genet. 19, 274–278. doi:10.1038/961 Ladi, E., Nichols, J.T., Ge, W., Miyamoto, A., Yao, C., Yang, L.T., Boulter, J., Sun, Y.E., Kintner, C., Weinmaster, G., 2005. The divergent DSL ligand Dll3 does not activate Notch signaling but cell autonomously attenuates signaling induced by other DSL ligands. J. Cell Biol. 170, 983–992. doi:10.1083/jcb.200503113 Lanford, P.J., Lan, Y., Jiang, R., Lindsell, C., Weinmaster, G., Gridley, T., Kelley, M.W., 1999. Notch signalling pathway mediates hair cell development in mammalian cochlea. Nat Genet 21, 289–292. doi:10.1038/6804 Lardelli, M., Dahlstrand, J., Lendahl, U., 1994. The novel Notch homologue mouse Notch 3 lacks specific epidermal growth factor-repeats and is expressed in proliferating neuroepithelium. Mech. Dev. 46, 123–136. doi:10.1016/0925-4773(94)90081-7 Laufer, E., Dahn, R., Orozco, O.E., Yeo, C.-Y., Pisenti, J., Henrique, D., Abbott, U.K., Fallon, J.F., Tabin, C., 1997. Expression of Radical fringe in limb-bud ectoderm regulates apical ectodermal ridge formation. Nature 386, 366–373. doi:10.1038/386366a0 36

Laurence, M.-K., Shergill, B., Kuon, J., Botvinick, E., Weinmaster, G., 2012. Notch Ligand Endocytosis Generates Mechanical Pulling Force Dependent on Dynamin, Epsins, and Actin. Dev. Cell 22, 1299–1312. doi:10.1016/j.devcel.2012.04.005 LaVoie, M.J., Selkoe, D.J., 2003. The Notch Ligands, Jagged and Delta, Are Sequentially Processed by ??-Secretase and Presenilin/??-Secretase and Release Signaling Fragments. J. Biol. Chem. 278, 34427–34437. doi:10.1074/jbc.M302659200 LeBon, L., Lee, T. V, Sprinzak, D., Jafar-Nejad, H., Elowitz, M.B., 2014. Fringe proteins modulate Notch-ligand cis and trans interactions to specify signaling states. Elife 3, e02950. doi:10.7554/eLife.02950 Lehner, B., Crombie, C., Tischler, J., Fortunato, A., Fraser, A.G., 2006. Systematic mapping of genetic interactions in Caenorhabditis elegans identifies common modifiers of diverse signaling pathways. Nat. Genet. 38, 896–903. doi:10.1038/ng1844 Lewis, A.K., Frantz, G.D., Carpenter, D.A., de Sauvage, F.J., Gao, W.-Q., 1998. Distinct expression patterns of notch family receptors and ligands during development of the mammalian inner ear. Mech. Dev. 78, 159–163. doi:10.1016/S0925-4773(98)00165-8 Li, X.Y., Zhai, W.J., Teng, C.B., 2015. Notch signaling in pancreatic development. Int. J. Mol. Sci. doi:10.3390/ijms17010048 Liu, Z., Brunskill, E., Varnum-Finney, B., Zhang, C., Zhang, A., Jay, P.Y., Bernstein, I., Morimoto, M., Kopan, R., 2015. The intracellular domains of Notch1 and Notch2 are functionally equivalent during development and carcinogenesis. Development 142, 2452–63. doi:10.1242/dev.125492 Liu, Z., Chen, S., Boyle, S., Zhu, Y., Zhang, A., Piwnica-Worms, D., Ilagan, M.X., Kopan, R., 2013. The extracellular domain of notch2 increases its cell-surface abundance and ligand responsiveness during kidney development. Dev. Cell 25, 585–598. 37

doi:10.1016/j.devcel.2013.05.022 Maass, J.C., Gu, R., Basch, M.L., Waldhaus, J., Lopez, E.M., Xia, A., Oghalai, J.S., Heller, S., Groves, A.K., 2015. Changes in the regulation of the Notch signaling pathway are temporally correlated with regenerative failure in the mouse cochlea. Front. Cell. Neurosci. 9, 110. doi:10.3389/fncel.2015.00110 Manderfield, L.J., High, F.A., Engleka, K.A., Liu, F., Li, L., Rentschler, S., Epstein, J.A., 2012. Notch Activation of Jagged1 Contributes to the Assembly of the Arterial WallClinical Perspective. Circulation 125. Marklund, U., Hansson, E.M., Sundström, E., de Angelis, M.H., Przemeck, G.K.H., Lendahl, U., Muhr, J., Ericson, J., 2010. Domain-specific control of neurogenesis achieved through patterned regulation of Notch ligand expression. Development 137. Mašek, J., Andersson, E.R., 2017. The developmental biology of genetic Notch disorders. Development 144, 1743–1763. doi:10.1242/dev.148007 Matsuda, M., Koga, M., Woltjen, K., Nishida, E., Ebisuya, M., 2015. Synthetic lateral inhibition governs cell-type bifurcation with robust ratios. Nat. Commun. 6, 6195. doi:10.1038/ncomms7195 Moloney, D.J., Panin, V.M., Johnston, S.H., Chen, J., Shao, L., Wilson, R., Wang, Y., Stanley, P., Irvine, K.D., Haltiwanger, R.S., Vogt, T.F., 2000. Fringe is a glycosyltransferase that modifies Notch. Nature 406, 369–375. doi:10.1038/35019000 Moran, J.L., Levorse, J.M., Vogt, T.F., 1999. Limbs move beyond the Radical fringe. Nature 399, 742–743. doi:10.1038/21560 Moran, J.L., Shifley, E.T., Levorse, J.M., Mani, S., Ostmann, K., Perez-Balaguer, A., Walker, D.M., Vogt, T.F., Cole, S.E., 2009. Manic fringe is not required for embryonic development,

38

and fringe family members do not exhibit redundant functions in the axial skeleton, limb, or hindbrain. Dev. Dyn. 238, 1803–12. doi:10.1002/dvdy.21982 Morrison, A., Hodgetts, C., Gossler, A., Hrabé de Angelis, M., Lewis, J., 1999. Expression of Delta1 and Serrate1 (Jagged1) in the mouse inner ear. Mech. Dev. 84, 169–172. doi:10.1016/S0925-4773(99)00066-0 Müller, J., Rana, N.A., Serth, K., Kakuda, S., Haltiwanger, R.S., Gossler, A., 2014. Ofucosylation of the Notch Ligand mDLL1 by POFUT1 Is Dispensable for Ligand Function. PLoS One 9, e88571. doi:10.1371/journal.pone.0088571 Nakamasu, A., Takahashi, G., Kanbe, A., Kondo, S., 2009. Interactions between zebrafish pigment cells responsible for the generation of Turing patterns. Proc. Natl. Acad. Sci. U. S. A. 106, 8429–34. doi:10.1073/pnas.0808622106 Newman, A.P., White, J.G., Sternberg, P.W., 1995. The Caenorhabditis elegans lin-12 gene mediates induction of ventral uterine specialization by the anchor cell. Development 121, 263–71. Nigon, V.M., Félix, M.-A., 2017. History of research on C. elegans and other free-living nematodes as model organisms. WormBook 1–91. doi:10.1895/wormbook.1.181.1 Nobelprize.org., 2014. The Nobel Prize in Physiology or Medicine 2002 [WWW Document]. Nobel Media Ab. Ohyama, T., Groves, A.K., 2004. Generation of Pax2-Cre Mice by Modification of a Pax2 Bacterial Artificial Chromosome. Genesis 38, 195–199. doi:10.1002/gene.20017 Okubo, Y., Sugawara, T., Abe-Koduka, N., Kanno, J., Kimura, A., Saga, Y., 2012. Lfng regulates the synchronized oscillation of the mouse segmentation clock via trans-repression of Notch signalling. Nat. Commun. 3, 1141. doi:10.1038/ncomms2133

39

Ong, C.-T., Sedy, J.R., Murphy, K.M., Kopan, R., Quatrano, R., 2008. Notch and Presenilin Regulate Cellular Expansion and Cytokine Secretion but Cannot Instruct Th1/Th2 Fate Acquisition. PLoS One 3, e2823. doi:10.1371/journal.pone.0002823 Park, S.K., Choi, V.N., Hwang, B.J., 2013. LIN-12/notch regulates lag-1 and lin-12 expression during anchor cell/ventral uterine precursor cell fate specification. Mol. Cells 35, 249–254. doi:10.1007/s10059-013-2333-6 Parks, A.L., Klueg, K.M., Stout, J.R., Muskavitch, M.A., 2000. Ligand endocytosis drives receptor dissociation and activation in the Notch pathway. Development 127, 1373–85. doi:10.1038/ng0797-212 Parks, A.L., Stout, J.R., Shepard, S.B., Klueg, K.M., Dos Santos, A.A., Parody, T.R., Vaskova, M., Muskavitch, M.A.T., 2006. Structure-function analysis of delta trafficking, receptor binding and signaling in Drosophila. Genetics 174, 1947–61. doi:10.1534/genetics.106.061630 Petrovic, J., Formosa-Jordan, P., Luna-Escalante, J.C., Abello, G., Ibanes, M., Neves, J., Giraldez, F., 2014. Ligand-dependent Notch signaling strength orchestrates lateral induction and lateral inhibition in the developing inner ear. Development 141, 2313–2324. doi:10.1242/dev.108100 Pitsouli, C., Delidakis, C., 2005. The interplay between DSL proteins and ubiquitin ligases in Notch signaling. Development 132. Preuße, K., Tveriakhina, L., Schuster-Gossler, K., Gaspar, C., Rosa, A.I., Henrique, D., Gossler, A., Stauber, M., 2015. Context-Dependent Functional Divergence of the Notch Ligands DLL1 and DLL4 In Vivo. PLoS Genet. 11, e1005328. doi:10.1371/journal.pgen.1005328 Priess, J.R., 2005. Notch signaling in the C. elegans embryo. WormBook 1–16. doi:10.1895/wormbook.1.4.1 40

Rg Waldhaus, J., Durruthy-Durruthy, R., Heller Correspondence, S., 2015. Quantitative HighResolution Cellular Map of the Organ of Corti. CellReports 11, 1385–1399. doi:10.1016/j.celrep.2015.04.062 Rodriguez-Esteban, C., Schwabe, J.W.R., Peña, J.D. La, Foys, B., Eshelman, B., Belmonte, J.C.I., 1997. Radical fringe positions the apical ectodermal ridge at the dorsoventral boundary of the vertebrate limb. Nature 386, 360–366. doi:10.1038/386360a0 Ross, D.A., Kadesch, T., 2004. Consequences of Notch-mediated induction of Jagged1. Exp. Cell Res. 296, 173–82. doi:10.1016/j.yexcr.2004.02.003 Sanchez-Irizarry, C., Carpenter, A.C., Weng, A.P., Pear, W.S., Aster, J.C., Blacklow, S.C., 2004. Notch Subunit Heterodimerization and Prevention of Ligand-Independent Proteolytic Activation Depend, Respectively, on a Novel Domain and the LNR Repeats. Mol. Cell. Biol. 24, 9265–9273. doi:10.1128/MCB.24.21.9265-9273.2004 Sasaki, Y., Ishida, S., Morimoto, I., Yamashita, T., Kojima, T., Kihara, C., Tanaka, T., Imai, K., Nakamura, Y., Tokino, T., 2002. The p53 family member genes are involved in the Notch signal pathway. J. Biol. Chem. 277, 719–724. doi:10.1074/jbc.M108080200 Scheffer, D.I., Shen, J., Corey, D.P., Chen, Z.-Y., 2015. Gene Expression by Mouse Inner Ear Hair Cells during Development. J. Neurosci. 35, 6366–6380. doi:10.1523/JNEUROSCI.5126-14.2015 Schindler, A.J., Sherwood, D.R., 2013. Morphogenesis of the caenorhabditis elegans vulva. Wiley Interdiscip. Rev. Dev. Biol. 2, 75–95. doi:10.1002/wdev.87 Schweisguth, F., 2015. Asymmetric cell division in the Drosophila bristle lineage: from the polarization of sensory organ precursor cells to Notch-mediated binary fate decision. Wiley Interdiscip. Rev. Dev. Biol. 4, 299–309. doi:10.1002/wdev.175

41

Serth, K., Schuster-Gossler, K., Kremmer, E., Hansen, B., Marohn-Köhn, B., Gossler, A., 2015. O-Fucosylation of DLL3 Is Required for Its Function during Somitogenesis. PLoS One 10, e0123776. doi:10.1371/journal.pone.0123776 Seugnet, L., Simpson, P., Haenlin, M., 1997. Transcriptional regulation of Notch and Delta: requirement for neuroblast segregation in Drosophila. Development 124, 2015–2025. Shaya, O., Binshtok, U., Hersch, M., Rivkin, D., Weinreb, S., Amir-Zilberstein, L., Khamaisi, B., Oppenheim, O., Desai, R.A., Goodyear, R.J., Richardson, G.P., Chen, C.S., Sprinzak, D., 2017. Cell-Cell Contact Area Affects Notch Signaling and Notch-Dependent Patterning. Dev. Cell 40, 505–511.e6. doi:10.1016/j.devcel.2017.02.009 Sheldon, H., Heikamp, E., Turley, H., Dragovic, R., Thomas, P., Oon, C.E., Leek, R., Edelmann, M., Kessler, B., Sainson, R.C.A., Sargent, I., Li, J.-L., Harris, A.L., 2010. New mechanism for Notch signaling to endothelium at a distance by Delta-like 4 incorporation into exosomes. Blood 116. Shifley, E.T., Cole, S.E., 2008a. Lunatic fringe protein processing by proprotein convertases may contribute to the short protein half-life in the segmentation clock. Biochim. Biophys. Acta Mol. Cell Res. 1783, 2384–2390. doi:10.1016/j.bbamcr.2008.07.009 Shifley, E.T., Cole, S.E., 2008b. Lunatic fringe protein processing by proprotein convertases may contribute to the short protein half-life in the segmentation clock. Biochim. Biophys. Acta Mol. Cell Res. 1783, 2384–2390. doi:10.1016/j.bbamcr.2008.07.009 Shimizu, K., Chiba, S., Kumano, K., Hosoya, N., Takahashi, T., Kanda, Y., Hamada, Y., Yazaki, Y., Hirai, H., 1999. Mouse Jagged1 physically interacts with Notch2 and other Notch receptors. Assessment by quantitative methods. J. Biol. Chem. 274, 32961–32969. doi:10.1074/jbc.274.46.32961 Shimizu, K., Chiba, S., Saito, T., Kumano, K., Takahashi, T., Hirai, H., 2001. Manic fringe and 42

lunatic fringe modify different sites of the Notch2 extracellular region, resulting in different signaling modulation. J. Biol. Chem. 276, 25753–8. doi:10.1074/jbc.M103473200 Shimojo, H., Kageyama, R., 2016. Oscillatory control of Delta-like1 in somitogenesis and neurogenesis: A unified model for different oscillatory dynamics. Semin. Cell Dev. Biol. 49, 76–82. doi:10.1016/j.semcdb.2016.01.017 Sjöqvist, M., Antfolk, D., Ferraris, S., Rraklli, V., Haga, C., Antila, C., Mutvei, A., Imanishi, S.Y., Holmberg, J., Jin, S., Eriksson, J.E., Lendahl, U., Sahlgren, C., 2014. PKCζ regulates Notch receptor routing and activity in a Notch signaling-dependent manner. Cell Res. 24, 433–50. doi:10.1038/cr.2014.34 Sprinzak, D., Lakhanpal, A., LeBon, L., Santat, L.A., Fontes, M.E., Anderson, G.A., GarciaOjalvo, J., Elowitz, M.B., 2010. Cis-interactions between Notch and Delta generate mutually exclusive signalling states. Nature 465, 86–90. doi:10.1038/nature08959 Stephenson, N.L., Avis, J.M., 2012. Direct observation of proteolytic cleavage at the S2 site upon forced unfolding of the Notch negative regulatory region. Pnas 109, E2757–E2765. doi:10.1073/pnas.1205788109 Sternberg, P.W., 2017. In Retrospect: Forty years of cellular clues from worms. Nature 543, 628–630. doi:10.1038/543628a Sternberg, P.W., 2005. Vulval development. WormBook 1–28. doi:10.1895/wormbook.1.6.1 Sternberg, P.W., 1988. Lateral inhibition during vulval induction in Caenorhabditis elegans. Nature 335, 551–554. doi:10.1038/335551a0 Sternberg, P.W., Horvitz, H.R., 1989. The combined action of two intercellular signaling pathways specifies three cell fates during vulval induction in C. elegans. Cell 58, 679–693. doi:10.1016/0092-8674(89)90103-7

43

Struhl, G., Fitzgerald, K., Greenwald, I., 1993. Intrinsic activity of the lin-12 and Notch intracellular domains in vivo. Cell 74, 331–345. doi:10.1016/0092-8674(93)90424-O Sulston, J.E., Brenner, S., 1974. The DNA of Caenorhabditis elegans. Genetics 77, 95–104. Sulston, J.E., Horvitz, H.R., 1977. Post-embryonic cell lineages of the nematode, Caenorhabditis elegans. Dev. Biol. 56, 110–56. Tamura, K., Taniguchi, Y., Minoguchi, S., Sakai, T., Tun, T., Furukawa, T., Honjo, T., 1995. Physical interaction between a novel domain of the receptor Notch and the transcription factor RBP-Jκ/Su(H). Curr. Biol. 5, 1416–1423. doi:10.1016/S0960-9822(95)00279-X Taylor, P., Takeuchi, H., Sheppard, D., Chillakuri, C., Lea, S.M., Haltiwanger, R.S., Handford, P.A., 2014. Fringe-mediated extension of O-linked fucose in the ligand-binding region of Notch1 increases binding to mammalian Notch ligands. Proc. Natl. Acad. Sci. 111, 7290– 7295. doi:10.1073/pnas.1319683111 Troost, T., Schneider, M., Klein, T., Schneider, M., Bardin, A., 2015. A Re-examination of the Selection of the Sensory Organ Precursor of the Bristle Sensilla of Drosophila melanogaster. PLoS Genet. 11, e1004911. doi:10.1371/journal.pgen.1004911 Turing, A.M., 1952. The Chemical Basis of Morphogenesis. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 237, 37–72. Uyttendaele, H., Marazzi, G., Wu, G., Yan, Q., Sassoon, D., Kitajewski, J., 1996. Notch4/int-3, a mammary proto-oncogene, is an endothelial cell-specific mammalian Notch gene. Development 122. Van de Walle, I., De Smet, G., Gärtner, M., De Smedt, M., Waegemans, E., Vandekerckhove, B., Leclercq, G., Plum, J., Aster, J.C., Bernstein, I.D., Guidos, C.J., Kyewski, B., Taghon, T., 2011. Jagged2 acts as a Delta-like Notch ligand during early hematopoietic cell fate

44

decisions. Blood 117. Van de Walle, I., Waegemans, E., De Medts, J., De Smet, G., De Smedt, M., Snauwaert, S., Vandekerckhove, B., Kerre, T., Leclercq, G., Plum, J., Gridley, T., Wang, T., Koch, U., Radtke, F., Taghon, T., 2013. Specific Notch receptor-ligand interactions control human TCR-αβ/γδ development by inducing differential Notch signal strength. J. Exp. Med. 210, 683–97. doi:10.1084/jem.20121798 Venkatraman, L., Regan, E.R., Bentley, K., 2016. Time to Decide? Dynamical Analysis Predicts Partial Tip/Stalk Patterning States Arise during Angiogenesis. PLoS One 11, e0166489. doi:10.1371/journal.pone.0166489 Wilkinson, H.A., Fitzgerald, K., Greenwald, I., 1994. Reciprocal changes in expression of the receptor lin-12 and its ligand lag-2 prior to commitment in a C. elegans cell fate decision. Cell 79, 1187–98. doi:10.1016/0092-8674(94)90010-8 Wolpert, L., 1969. Positional information and the spatial pattern of cellular differentiation. J. Theor. Biol. 25, 1–47. Wu, J.Y., Rao, Y., 1999. Fringe: defining borders by regulating the Notch pathway. Curr. Opin. Neurobiol. 9, 537–543. doi:10.1016/S0959-4388(99)00020-3 Yang, L.T., Nichols, J.T., Yao, C., Manilay, J.O., Robey, E.A., Weinmaster, G., 2005. Fringe glycosyltransferases differentially modulate Notch1 proteolysis induced by Delta1 and Jagged1. Mol Biol Cell 16, 927–942. doi:10.1091/mbc.E04-07-0614 Yuan, Y.P., Schultz, J., Mlodzik, M., Bork, P., 1997. Secreted Fringe-like Signaling Molecules May Be Glycosyltransferases. Cell 88, 9–11. doi:10.1016/S0092-8674(00)81852-8 Yuet, K.P., Doma, M.K., Ngo, J.T., Sweredoski, M.J., Graham, R.L.J., Moradian, A., Hess, S., Schuman, E.M., Sternberg, P.W., Tirrell, D.A., 2015. Cell-specific proteomic analysis in

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Caenorhabditis elegans. Proc. Natl. Acad. Sci. 112, 2705–2710. doi:10.1073/pnas.1421567112 Zhang, N., Gridley, T., 1998. Defects in somite formation in lunatic fringe-deficient mice. Nature 394, 374–377. doi:10.1038/28625 Zhang, N., Martin, G. V., Kelley, M.W., Gridley, T., 2000. A mutation in the Lunatic fringe gene suppresses the effects of a Jagged2 mutation on inner hair cell development in the cochlea. Curr. Biol. 10, 659–662. doi:10.1016/S0960-9822(00)00522-4 Öberg, C., Li, J., Pauley, A., Wolf, E., Gurney, M., Lendahl, U., 2001. The Notch Intracellular Domain Is Ubiquitinated and Negatively Regulated by the Mammalian Sel-10 Homolog. J. Biol. Chem. 276, 35847–35853. doi:10.1074/jbc.M103992200

Table 1. Effects of Fringe-mediated Notch Receptor modifications on signaling induced by specific Notch ligands.

Dll1 Lfng

Notch1

Mfng

Rfng

+ (Kakuda and Haltiwanger, 2017; LeBon et al., 2014; Yang et al., 2005) + (Kakuda and Haltiwanger, 2017; LeBon et al., 2014; Yang et al., 2005) + (Kakuda and Haltiwanger, 2017; LeBon et al., 2014; Yang et al., 2005)

Jag1 (Abe et al., 2010; Hicks et al., 2000; Kakuda and Haltiwanger, 2017; LeBon et al., 2014; Shifley and Cole, 2008a; Yang et al., 2005) (Benedito et al., 2009; D’Amato et al., 2015; Hicks et al., 2000; Kakuda and Haltiwanger, 2017; LeBon et al., 2014; Yang et al., 2005) + (Kakuda and Haltiwanger, 2017; LeBon et al., 2014; Shifley and Cole, 2008b; Yang et al., 2005)

46

Dll4

Jag2

+ (Abe et al., 2010)

+/(Abe et al., 2010; Van de Walle et al., 2011)

+ (Benedito et al., 2009; D’Amato et al., 2015)

(D’Amato et al., 2015)

Notch2

Lfng

Mfng

+ (Hicks et al., 2000)

+/(Hicks et al., 2000; Shimizu et al., 2001)

+ (Shimizu et al., 2001)

(Shimizu et al., 2001)

Rfng

Table 2. Examples of lateral inhibition and lateral induction processes in vivo Organism

Lateral Inhibition Process

Reference

Caenorhabditis Vulval and uterine development employs a For review, see (Schindler elegans

series

of

lateral

inhibition

events

to and Sherwood, 2013) and

coordinate anchor cell selection, repression text. of primary cell fates in vulval precursor cells, and inhibition of the primary fate in ventral uterine cells. See also Fig 3. Drosophila

During the formation of epidermal sensory For review see

melanogaster

bristles, Notch-mediated lateral inhibition See also (Corson et al., acts to first induce a proneural stripe of 2017) competence, and thereafter singles out sensory organ precursors that give rise to mechanosensory bristles.

Danio rerio

During zebrafish primary neurogenesis, deltaA, deltaB and deltaD mediate lateral inhibition to select primary neurons.

Mus musculus During (mammals)

inner

ear

development

lateral General

induction by Jag1 originate prosensory development 47

inner

ear

and

Notch

patches. Hair cell inducing signals give rise signaling therein (Martin L. to Dll1 and Jag2 expressing hair cell Basch et al., 2016; Kiernan, progenitors, which produce a pattern of hair 2013) and

supporting

cells

through

lateral

inhibition. See also Fig 4 During

vascular

smooth

muscle

cell (Manderfield et al., 2012)

(VSMC) development on arteries, lateral induction by Jag1 on endothelial cells induces neural crest differentiation into VSMCs, which then instructs further layers of neural crest to differentiate into VSMCs. During pancreatic development, lateral (Li et al., 2015) inhibition

regulates

differentiation

of

endocrine cells.

Fig 1. Notch signaling mediates lateral inhibition and lateral induction. (A) Notch signaling is initiated when a Notch ligand on a signal-sending cell (blue) binds to a Notch receptor on a contacting signal-receiving cell (orange). Binding results in proteolytic processing of the Notch reccptor, releasing the Notch intracellular domain (NICD) which translocates to the nucleus to activate signaling together with the transcription factor CSL (CBF-1/Su(H)/LAG1-1; aka RPJk) and co-acticator Mastermind-like (MAML). (B) Lateral inhibition is a process by which Notch

48

signaling inhibits neighbors in a pool of initially equivalent cells from taking on a default cell state. Notch receptor activation results in downregulation of ligand activation potential, either through decreased ligand expression or through Fringe modification (see text and Fig 2 for more detail). This mechanism amplifies stochastic differences in ligand or receptor expression, such that one cell becomes signal-sending and one is signal-receiving. (C) This typically results in patterns of cells with a signal-sending (blue) cells inhibiting several neighbors (orange) from adopting a default cell state. The range of inhibition may be increase by filopodia extensions. (C) Lateral induction is a process by which Notch signaling induces neighbors in a pool of initially equivalent cells to adapt a common fate. Notch receptor activation directly upregulates ligand expression in signal-receiving cells (purple), which can then activate Notch receptors in further contacting cells (purple). (D Lateral induction results in a wave of cells being induced to adapt the same fate.

Fig 2. Indirect and direct signaling mechanisms govern lateral inhibition and induction, respectively. (A) Lateral inhibition is mediated by down-regulation of Notch ligand activity in signal-receiving cells. Two non-mutually exclusive mechanisms contribute to dampened ligand activity. NICD/CSL transcription complexes induce expression of Notch target genes, of the enhancer of Split [E(spl)] complex, that then repress expression of Achaete-Scute (Ac-Sc) genes. These neurogenic genes would otherwise maintain ligand expression. For example, in C.elegans anchor cell selection, LIN-12 activation by LAG-2 downregulates LAG-2 transcription via downregulation of HLH-2 (Karp and Greenwald, 2003). Another mode of lateral inhibition is mediated by Fringe, whose expression in signal-sending cells potentiates trans-activation potential, or discriminates between different ligands, and expression in signal-receiving cells increases ligand-mediated cis-inhibtion. (B) Lateral induction results in propagation of upregulation of Notch ligand in signal-receiving cells. In this case, Notch ligand, for example Jag1, is a direct target of Notch signaling. 49

Fig 3. Lateral cues regulate multiple steps in C. elegans vulval and uterine development. The anchor cell (AC) is the master regulator of vulval development. (A) The anchor cell arises from lateral inhibition between two equivalent alpha cells, in which stoichiometric differences in receptor and ligand expression are amplified by negative feedback, such that the LAG-2expressing cell inhibits the LIN-12/Notch-expressing cell from adapting the anchor cell fate. The LIN-12/Notch-expressing cell instead adopts a ventral uterine (VU) cell fate, while the LAG-2expressing cell becomes the anchor cell. (B) The anchor cell secretes LIN-3/EGF which induces a 1° cell fate in P6p, one of the adjacent vulval precursor cells (VPCs, P3p-P8p). The 1° fate VPC expresses DSL, which inhibits adjacent VPCs from adopting the 1° fate, and also induces the 2° fate. (C) Finally, the LAG-2 expressing anchor cell inhibits 6 adjacent ventral uterine cells (grey) from adopting the default rho state, causing them to instead differentiate into pi (π) cells.

Fig 4.The organ of Corti, in the inner ear, is a biological model that integrates both lateral induction and lateral inhibition processes during its development. Overview and panel 4. Inner hair cells convert mechanical stimuli in the form of soundwaves into electrical nerve impulses transmitted to the auditory cortex. Outer hair cells, on the other hand, have a signalamplifying function. Deiters’ cells and inner phalangeal cells range from the basilar membrane to the reticular lamina and provide support for the outer and inner hair cells, respectively. Pillar cells are highly specialized cells creating the tunnel of Corti, and separating the inner and outer hair cells. (1) Jag1 expression mediates prosensory domain formation via lateral induction. Next (2) Jag1 and Jag2 expression adjacent to Kölliker’s organ represses cells in that domain to adopt an organ of Corti fate, establishing a precise boundary for organ of Corti formation. (3) Jag2 and Dll1 expression, via lateral inhibition, repress adjacent cells from adopting a hair cell fate. The

50

repressed cells instead become different types of supporting cells, resulting in the tightly regulated final structure of the organ of Corti (4).

HIGHLIGHTS

-

Lateral patterning mediate precise spatial cues during development. Notch signaling drives both lateral inhibition and lateral induction patterning. Inhibitive cues prevent cells adopting default states. Inductive cues prompt adoption of specific cell fates. Here, we discuss examples of Notch lateral signaling in flies, worms and mammals.

51

B Lateral inhibition

NICD MAML

CSL

E Lateral induction

Direction of activation

C Lateral inhibition

D Lateral induction

Notch ON Signal receiving & signal -sending

Notch ligand Notch receptor

Notch ON Notch OFF signal receiving Signal-sending

A Notch signaling

Sjöqvist and Andersson, Figure 1

B. Lateral induction Signal-sending

Signal-sending

A. Lateral inhibition

2 LFng E(spl) genes Ac-Sc genes

Notch ligand

Notch ON signal receiving

Notch ON signal receiving

1

Sjöqvist and Andersson, Figure 2

Notch Notch = ligand target gene

C. elegans Vulva A Anchor cell selection

B

C

Vulval development

Vulval uterine connection LIN-12 / Notch LAG-2

α

α

A AC C

AC A C LIN-12 / Notch

LAG-2

P3p

P4p

P5p

P6p

P7p

P8p

3°

3°

2°

1°

2°

3°

2°

3°

Time

AC AC LIN-3/EGF

VU

AC

3°

3°

2°

1°

π π LIN-12 / Notch

2°

3°

DSL Induction of 2° fate Repression of 1° fate

Sjöqvist & Andersson Fig 3

3°

3°

2°

π A AC C π π π 1°

Cochlear duct Vestibular canal Tectorial membrane Inner ear

Organ of Corti Tympanic canal

1

Prosensory domain formation

2

Jag1-mediated lateral induction

Boundary establishment

Jag1/Jag2-mediated lateral inhibition establishes a boundary towards Kölliker’s organ

4

3

Inner hair cell

Jag2- & Dll1- mediated lateral inhibition represses supporting cells from adopting hair cell fate

Pillar cells

Phalangeal Tunnel of Corti cells

Sjöqvist and Andersson, Figure 4

Outer hair cells

Deiters’ cells