outstanding questions in developmental erk signaling · review outstanding questions in...

REVIEW

Outstanding questions in developmental ERK signalingAleena L. Patel and Stanislav Y. Shvartsman*

ABSTRACTThe extracellular signal-regulated kinase (ERK) pathway leads toactivation of the effector molecule ERK, which controls downstreamresponses by phosphorylating a variety of substrates, includingtranscription factors. Crucial insights into the regulation and function ofthis pathway came from studying embryos in which specific phenotypesarise fromaberrant ERKactivation. Despite decadesof research, severalimportant questions remain to be addressed for deeper understandingof this highly conserved signaling system and its function. Answeringthese questions will require quantifying the first steps of pathwayactivation, elucidating the mechanisms of transcriptional interpretationand measuring the quantitative limits of ERK signaling within whichthe system must operate to avoid developmental defects.

KEY WORDS: Inductive ERK signaling, Quantitative parameters,Regulatory networks, Transcriptional interpretation

IntroductionAnimal development relies on a small set of signaling systems actingin combination to guide pattern formation and tissue morphogenesis(Martinez-Arias and Stewart, 2002). By now we have a nearlycomplete parts lists of at least the core elements of these systems andare studying them at multiple levels of biological organization.However, we are still far from understanding what makes signalingsystems robust and how a single pathway can have such diverseoutputs, and also from being able to explain how relatively subtleperturbations to signaling transduction can cause developmentalabnormalities (Tidyman and Rauen, 2012; Rauen, 2013). Here, wefocus on the extracellular signal-regulated kinase (ERK) cascade, anessential regulator of animal development (Fig. 1) (Gabay et al., 1997;Dorey andAmaya, 2010; Corson et al., 2003). Using three extensivelystudied experimental models of developmental ERK signaling, wehighlight some of the key outstanding questions that must beaddressed to achieve the next level of understanding. In each of thesemodels, ERK signaling is triggered by a well-defined ligand sourceand, via an intracellular phosphorylation cascade, induces spatialpatterns of gene expression in a field of responding cells. Althoughthis scenario is certainly not the only mode of developmental ERKsignaling (Molotkov et al., 2017; Kang et al., 2017; Reim et al., 2012;Kadam et al., 2012; Stathopoulos et al., 2004), its relative simplicitymakes it especially attractive for discussing the most crucialunanswered questions. These questions, and the insights we cangain into them from simple systems, should also be relevant to morecomplex scenarios.We start by discussing unanswered questions related to the

processes at the input layer of the ERK cascade, focusing onthe spatiotemporal control of receptor activation. We then turn to

the transcriptional interpretation of ERK activation. The followingsection focuses on the mechanisms that ensure robust signaling anddiscusses the origins of ERK-dependent developmental defects. Weclose by proposing directions for future studies and discuss therelevance of the stated questions for other signaling systems.

Key unanswered questions and model systems for theiranalysisAs summarized above, we focus here on three major areas where westill have much to learn about the ERK pathway and its effects. Thefirst set of questions is related to the quantitative understanding ofmechanisms that are alreadywell studied at the molecular and cellularlevels, such as signal initiation –when ligands bind to transmembranereceptor tyrosine kinases (RTKs) (Lemmon and Schlessinger, 2010).Despite decades of study, we still have a poor understanding of howthe absolute concentrations of ligand and receptor, and the kinetics oftheir interactions, impact both quantitative and qualitative aspects ofsignal output. How many ligand-receptor complexes are required toinitiate a signal? How are ligand-receptor complexes spatiallydistributed in a field of responding cells? We therefore need to beable to quantify the numbers of active RTKs required to triggerintracellular pathways to provide an absolute measure of signalinginputs. These numbers can be readily estimated in cultured cells;however, to the best of our knowledge, they have yet to be obtained ina single developmental context (Schoeberl et al., 2002; Stockmannet al., 2017).

The second set of questions addresses the mechanisms by whichactive ERK controls gene expression to influence developmentalpattern formation. In comparison with studies of ERK activation byupstream components of the pathway, the mechanisms by whichactive ERK alters the activities of downstream transcription factorsand basal transcription machinery are relatively unexplored (Kimet al., 2011; Kolch, 2000; Hollenhorst et al., 2011). What physicalchanges to transcription factors are induced by ERK activity? Wheredo these changes occur in the cell?We need a better understanding ofhow the ERK pathway regulates transcriptional activators andrepressors to alter the gene expression profile of a cell – and how itcan induce diverse transcriptional outputs in different contexts.

Finally, a third set of questions is motivated by studies of a largeclass of genetically based human developmental abnormalitiesassociated with deregulated ERK signaling (reviewed by Jindalet al., 2015). Although it is believed that these abnormalities reflectquantitative changes in the spatiotemporal distributions ofdevelopmental ERK signals, the magnitudes of pertubation areessentially unknown and we have a poor understanding of the limitswithin which the pathway must operate to achieve normaldevelopment (Goyal et al., 2017). What are the maximum andminimum amplitudes and durations of ERK activity that still lead tonormal patterning outcomes? To what extent must ERK activity berestricted in space? Quantifying these parameters is necessary forprobing the intrinsic robustness of developmental ERK signals.

We discuss these questions in the context of the threewell-studiedinductive events in Ciona intestinalis, Drosophila melanogaster

Lewis Sigler Institute for Integrative Genomics, Department of ChemicalEngineering, Princeton University, Princeton, NJ 08544, USA.

*Author for correspondence ([email protected])

S.Y.S., 0000-0002-9152-9334

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and Caenorhabditis elegans. In the 32-cell stage Ciona embryo,fibroblast growth factor (FGF) is secreted from 16 vegetal cells andactivates the ERK pathway in immediately adjacent animal cells(Fig. 2A) (Bertrand et al., 2003). The downstream target of the ERKpathway, the neural marker otx, is consequently expressed in a four-cell pattern in the animal hemisphere – specifically in the a6.5 andb6.5 pairs of cells that have the highest surface contact with thevegetal cells (Hudson and Lemaire, 2001; Tassy et al., 2006). otxultimately specifies part of the neural lineage of the Ciona embryo(Wada et al., 2002; Imai et al., 2006). ERK activity also specifiespart of the neural lineage in the early Drosophila embryo. In thiscase, two ventrolateral stripes of the epidermal growth factor (EGF)in the 3 h-old syncytial embryo activate the ERK pathway in anautocrine manner (discussed further below), inducing expression ofits downstream target: the homeobox transcription factor ind(Fig. 2B) (Lim et al., 2015; Von Ohlen and Doe, 2000). In thefinal example, a gradient of EGF ligand, released from a single cell,induces a stereotypic cell fate pattern in the underlying epidermisthat always includes a single cell bearing primary (1°) fate in thevulval precursor cells (VPCs) of the C. elegans larva (Fig. 2C)(Katz et al., 1995). Each of these systems exhibits clear loss-of-function phenotypes in the absence of ERK activation: in Ciona and

Drosophila, the nervous system does not develop properly withoutERK signaling, while inC. elegans, insufficient ERK signaling leadsto a ‘vulvaless’ phenotype – the worm cannot lay eggs (Moghal andSternberg, 2003). Together, these three examples have served assignificant insights into how ERK signaling functions, and providevaluable testing grounds to further probe the questions laid out above.

Quantitative aspects of signal initiationERK signaling is initiated by ligand/receptor binding at the cellsurface (Fig. 1). In all three examples chosen for this Review, locallyproduced ligands reach their target receptors either by diffusingaway from the source (paracrine signaling; Fig. 3A), or by acting atshort range with diffusion being either insignificant or nonexistent( juxtacrine or autocrine signaling; Fig. 3B,C). We still have anincomplete understanding of what defines ligand diffusivity indifferent contexts, but it is clear that ligand range and concentrationwill be important determinants of signal response.

Paracrine signalingThe patterning of vulval precursor cells (VPCs) in C. elegansprovides what seems to be a case of pathway activation by diffusibleligands (Fig. 3A). In this system, the anchor cell (AC) secretes adiffusible ligand, EGF, towards the undifferentiated vulval precursorcells (VPC), named P3.p to P8.p (Moghal and Sternberg, 2003;Sternberg, 2005). EGF seems to activate the ERK pathway such thatthree distinct cell fates, primary (1°), secondary (2°) or tertiary (3°)are induced in a distance-dependent manner (Katz et al., 1995). Thegradient of ERK activation normally peaks at the VPC situatedclosest to the AC (P6.p), which is the VPC that always assumes 1°fate while the cells adjacent to P6.p take on 2° fate. The remainingperipheral cells become 3° cells (Sternberg and Horvitz, 1986).Although EGF forms a gradient, VPC induction may actuallyoccur sequentially such that P6.p is induced with 1° fate first,before inducing 2° fate in the VPCs adjacent to P6.p (Simske andKirn, 1995). EGF secreted by the AC induces expression of therhomboid protease ROM-1 in proximal VPCs, which then cleavesand activates a variant of EGF to which more distal cells aresensitive (Dutt et al., 2004). Signal relay from proximal to distalcells also increases the range of EGF.

The distribution of receptors available to bind and sense theextracellular EGF on the membranes of the VPCs will also controlthe ERK inputs. In VPCs, EGF receptor (EGFR) localization andmobility modulates the number of ligand-receptor complexesformed. For one, localization complexes target EGFR to thebasolateral membranes of VPCs, which face the EGF-secreting ACs(Simske et al., 1996; Kaech et al., 1998). Moreover, once targeted tothe basolateral membrane, an actin-binding protein, ERM1, cansequester EGFR in an inactive compartment. It is thought thatattachment to the cortical F-actin via ERM1 restricts EGFRmobilityand therefore its access to other proteins that are required forreceptor activation. This sequestration of a pool of EGFR permitslong-lasting sensing of the external EGF gradient during the courseof VPC induction (Haag et al., 2014).

The responses of the VPC pattern to variations in the strength ofsignaling inputs are inconsistent with the idea that minimumthresholds of ERK activation define cell fate. For example, partiallyreducing EGFR expression can lead to multiple VPCs with 1° fate,which results in a multivulva phenotype (Aroian and Sternberg,1991). Reduced signaling levels would not lead to more 1° fateinduction if a minimum threshold of ERK activity were required.A threshold model for cell fate induction suggests that the externalligand gradient serves as a morphogen, providing positional cues for

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Fig. 1. Simplified schematic of the ERK pathway. Growth factors activatethe ERK pathway by binding to transmembrane receptor tyrosine kinases(RTKs). The RTKs signal to the membrane-tethered GTPase Ras, which thenactivates the core phosphorylation cascade from the kinase Raf to mitogen-activated protein kinase kinase (MAP2K or MEK), to extracellular signal-regulated kinase (ERK). ERK can translocate to the nucleus, where it interactswith transcription factors to regulate target gene expression.

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cell fate among the VPCs. When the source of available ligand isincreased, this model would predict that more cells should beinduced with 1° fate. Instead, the wild-type 3°-3°-2°-1°-2°-3°pattern is maintained over an approximately threefold range of EGFlevels (Barkoulas et al., 2013). This robustness is in stark contrast toan analogous example of paracrine ERK signaling the Drosophilaoocyte, which is highly sensitive to the inductive ligand gradient.In this system, extra genetic doses of EGF are not tolerated and

strongly dorsalize the Drosophila egg (Neuman-Silberberg andSchupbach, 1994).

As discussed below, the emerging patterns of cell fates can beattributed to a variety of regulatory feedback mechanisms. Inaddition, a first level of control – ensuring specification of exactlyone cell with 1° fate – could be provided by the rapid sequestrationof the diffusible EGF ligand by P6.p. Such a model is reminiscent ofother systems involving long-range induction by a diffusible

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Fig. 2. Target gene expression in three modelsystems. Localized ERK pathway ligands (red) inducestereotypic patterns of target gene expression (blue) inthe three model organisms. (A) The 32-cellC. intestinalisembryo consists of 16 animal (An) and 16 vegetal (Vg)cells that are symmetric about the anterior-posterior (A-P) axis. Each symmetric cell pair has a unique name,with lowercase letters indicating cells in the animalhemisphere, and uppercase letters indicating cells in thevegetal hemisphere. otx (blue) is induced in exactly fourcells named the a6.5 and b6.5 pairs in the animalhemisphere of the embryo (Bertrand et al., 2003;Lemaire et al., 2002). Vegetal cells (red) produce FGFligands that induce otx via the ERK pathway in theneighboring animal cells. The area of contact betweenan animal cell and the vegetal cells adjacent to itdetermines the total amount of FGF ligand providingsignals to that cell. At this stage, the a6.5 and b6.5 pairshave the highest surface contact areas with vegetal cells,and therefore exhibit sufficient ERK activity to induce otxexpression. The surface contacts schematic is based onthe cell contact measurements provided for Cionaintestinalis type A on www.aniseed.cnrs.fr. (B) ind (blue)is expressed in ventrolateral stripes along the anterior-posterior (A-P) axis in theD. melanogaster embryo at 3 hpost fertilization, which at this stage is a syncytium withnuclei (gray) lining the periphery. This expression patternreflects localized ligand (EGF) production (red) also inthe ventrolateral domain of the embryo (Lim et al., 2015).Prepatterned transcription factors further limit the domainof ind expression. The left image shows a cross-sectionalong the A-P axis; the right image shows a cross-sectionalong the dorsal-ventral (D-V) axis. (C) One of sixequivalent vulval precursor cells (VPC) gives rise to avulva in C. elegans at the L2 stage. An anchor cell (AC,red) secretes EGF ligand and is positioned closest toP6.p, which is induced to generate a single cell with 1°fate (blue, marked by expression of lin-39 – an ERKtarget gene). The neighboring cells assume 2° cell fate(green). The three cells farthest from the EGF sourceassume 3° fate (gray). This specific fate pattern, 3°-3°-2°-1°-2°-3°, is required for correct vulva morphogenesis(Moghal and Sternberg, 2003).

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morphogen, in which interactions between the ligands and theircognate receptors that lead to self-enhanced ligand degradation areimportant for generating robust patterns (Eldar et al., 2003). Indeed,induction of distal VPCs with 1° fate occurs only when cells closestto the AC are ablated, supporting the idea that the VPCs sensingEGF at its highest concentration are capturing ligand before it candiffuse away (Sternberg and Horvitz, 1986; Sulston and White,1980). When EGFR expression is partially reduced, less EGFsequestration by P6.p could lead to increased ligand diffusion tomore distal VPCs, explaining the apparent hypersensitivity inthese mutants (Hajnal et al., 1997). Quantitative understanding ofthese effects requires accurate measurements of the spatiotemporaldistribution of EGF/EGFR complexes as well as a framework forconnecting the information about input layer of the patterningnetwork to the emerging cell fate patterns.

Juxtracrine signalingIn contrast to the VPC patterning system, in which one cell secretesa diffusible ligand, multiple ligand-producing cells collectivelycontribute to the total ERK input that induces otx in a subset of cellsof the 32-cell stage C. intestinalis embryo. In this system, priorstages of asymmetric cell division result in a unique set of contactswith adjacent cells (Fig. 2A) (Ohta and Satou, 2013; Rothbächeret al., 2007). Asymmetric maternal factors both predispose theanimal hemisphere to express the transcription factors required forotx expression (Oda-Ishii et al., 2016; Bertrand et al., 2003;Rothbächer et al., 2007) and are responsible for FGF secretion fromthe vegetal hemisphere (Imai et al., 2002). In these embryos, theintercellular space is too small to permit significant ligand diffusionfrom the source to the responding cell, so direct cell-cell contactsbetween the plasma membranes of ligand-producing and-responding cells are needed for ERK activation (Tassy et al.,2006). As a consequence, the ERK inputs are contact dependent andcan be thought of as juxtacrine (Fig. 3B). The animal cells that havethe highest surface contact areawith the FGF-secreting vegetal cells,named the a6.5 and b6.5 pairs, always express otx (Tassy et al.,2006). It is still unclear, however, whether ligand secretion isuniform on all faces of an inducing cell, and whether receptors areevenly distributed on the plasma membranes of responding cells.Addressing these issues is difficult without the ability to monitorligand-receptor interactions, but such techniques will be required ifwe are to understand in detail the mechanisms underlying thedefined spatial pattern of ERK activation.

Autocrine signalingAlthough the VPC and otx patterning systems are examples ofligand molecules diffusing from one source cell to another, ligandscan also bind to receptors on the same cells that produce them(Fig. 3C). For example, during ind induction in the Drosophilaembryo, EGF signaling appears to work in an autocrine regime,whereby ligand-producing cells also express cognate receptors. Atthis point, cellularization is not complete and the Drosophilaembryo is still a syncytiumwith multiple nuclei lining the periphery.Ligands produced in the syncytial embryo are secreted into theperivitelline space, which surrounds the common plasma membranethat contains EGF receptors. A transcriptional circuit downstream ofthe well-characterized ventral-dorsal Dorsal (Dl) morphogengradient establishes a two-striped pattern of the expression ofRhomboid (Rho), an intracellular protease that controls the secretionof the EGF ligand Spitz (Spi). An incoherent feed-forward loop isestablished when Dl induces both Rho and a repressor of Rho,Snail (Rushlow and Shvartsman, 2012). Rho is therefore induced

A Paracrine

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Fig. 3. Three modes of ligand-receptor interactions. (A) Inductive signalscan come from ligands that diffuse away from a localized source (paracrinesignaling). As a consequence, a ligand gradient forms in the extracellularspace across the field of response. In the C. elegans VPC induction example,EGF secreted by the anchor cell can diffuse away from the source towardsdistal cells. How the distribution of signaling complexes evolves among theVPCs depends on ligand diffusion through the extracellular space andcapture by surface receptors. (B) When ligand diffusion in the extracellularspace is extremely limited, direct contacts between ligand-producing and-responding cells ( juxtacrine signaling) control the number of complexes thatdictate the signaling dose. Ligands do not diffuse in the extracellular space toreach distant target cells. For example, in the early Ciona embryo, tissuegeometry restricts ERK activation by source cells to the directly adjacenttarget cells. The surface area of membrane contacts between ligand-producing and -responding cells therefore appears to directly control the levelof ERK activity (Tassy et al., 2006). (C) Cells can also secrete ligands thatactivate receptors on their own surface (autocrine signaling). During indinduction in Drosophila, EGF ligand secreted in the ventrolateral domain ofthe embryo does not diffuse significantly, and binds to receptors on the samecells (Lim et al., 2015).

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only in lateral cells in which intermediate levels of Dl are strongenough to induce Rhowhereas Snail repression is minimal. Stronglyoverlapping spatial profiles of Rho expression and active duallyphosphorylated ERK (dpERK) suggest that diffusion of secretedligand is negligible, most likely reflecting capture of secretedligands by the very same cells that produce them (Lim et al., 2015).Notably, prepatterned transcription factors limit ind expression to asubset of cells within the domain of dpERK activity. Interestingly,both Spi and EGFR are controlled by Zelda (Zld), a uniformlyexpressed activator of early zygotic transcription (Liang et al.,2008). A mathematical model of these processes, based on theassumptions that signaling levels are proportional to the number ofligand-bound EGF receptors and that ligand diffusion is negligible,predicts that signaling levels should rise as the cube of time,measured from the onset of ligand production. The time-resolvedmeasurements of the levels of dpERK support this prediction,suggesting that the signaling cascade connecting cell-surfacereceptors and ERK activation indeed functions as a linear system(Lim et al., 2015).

Quantitative analysis of ligand-receptor complexesStudies of some of these systems have led to the formulation ofmathematical models that have ligand-receptor complexes as theirkey variables (Giurumescu et al., 2006). At the same time, theabsolute values of active complexes are currently unknown,preventing direct testing of model predictions. Several existing toolsmay enable quantitative analysis of ligand binding in a developingembryo. In particular, fluorescence correlation spectroscopy (FCS)of fluorescently tagged ligands can measure ligand concentrationsand diffusivities in vivo. This technique may be useful forquantifying ligand concentrations in systems in which the ERK-activating ligands diffuse away from a localized source. Forexample, FCS analysis of the FGF8 morphogen in the zebrafishembryo has been shown to allow quantification of localconcentrations of ligand with high precision (Yu et al., 2009).Monitoring complexes and ligand-receptor interactions could alsobe enabled by live imaging of quantum dot-labeled ligands andfluorescently tagged receptors (Lim et al., 2016). Quantum dotligands can be detected at the single nanoparticle level and havealready been used to study ligand binding and transport phenomenaduring RTK signaling in cells (Lidke et al., 2004). Such techniqueswill allow us to monitor the evolution of ligand-receptor complexes,and directly measure quantitative aspects of signal initiation such asthe lifetime and turnover rate of a complex at the membrane.Moreover, these experiments have the potential to reveal importantroles of receptor trafficking in regulating ERK activation in time andspace. As illustrated in the VPC induction example, receptorlocalization and mobility influence the duration of an ERK signal.When and where ligand-bound and unbound receptors are traffickedwithin a cell could therefore dramatically alter how a cell sensesexternal signaling cues in other systems.

How is ERK signaling interpreted transcriptionally?ERK controls gene expression by phosphorylating transcriptionfactors and components of the basal transcription machinery. Thereported effects on transcription factors are diverse and includepotentiation of the effects of existing activators and antagonism ofrepressor functions. During VPC patterning in C. elegans,phosphorylation by ERK induces structural changes in at leasttwo transcription factors involved in the regulation of lin-39, a keygene responsible for the 1° vulval fate (Clark et al., 1993;Wagmaister et al., 2006) (Fig. 4A). This gene is initially repressed

by a complex formed by the forkhead transcription factor LIN-31and the ETS transcription factor LIN-1, which represses lin-39 byinteracting with nucleosome remodeling and deacetylationcomplexes (Guerry et al., 2007; Maloof and Kenyon, 1998;Leight et al., 2005; Miller et al., 1993; Tan et al., 1998). ERKphosphorylates the C-terminal domain of LIN-1, disrupting therepressor complex and potentially turning LIN-1 into an activator oflin-39 (Fig. 4A, bottom) (Jacobs et al., 1998; Tiensuu et al., 2005;Leight et al., 2015; Wagmaister et al., 2006). The role of LIN-1 asan activator or a repressor depends on its phosphorylation status,and on the particular target gene. Although ERK-mediatedphosphorylation of LIN-1 converts it to an activator of 1° fategenes (Leight et al., 2015), for other target genes, LIN-1 may actsolely as a repressor. For example, transcription of lateral Notchligand genes (see below) does not require LIN-1 activation(Underwood et al., 2017). Furthermore, dissociation of theLIN-1/LIN-31 complex exposes a phosphorylation site in thetransactivation domain of LIN-31 such that it also becomes anactivator when phosphorylated by ERK (Fig. 4A) (Tan et al.,1998). In the current model of the 1° vulval fate induction, activeERK first disrupts the LIN-1/LIN-31 complex that repressesthe key target gene, and then converts one or both componentsof this complex into a direct activator of the same gene (Tan et al.,1998; Sundaram, 2013). Thus, ERK signaling both relievesrepression of a target gene and promotes its activation, as if firstreleasing the brakes of a car and then pressing on the accelerator.This model is not unique to the specific example of VPC inductionin C. elegans. In Drosophila, the ETS factors Pointed (Pnt)and Yan collaboratively repress target gene transcription. Geneexpression is induced when an ERK signal leads to disruption ofthe repressive complex followed by Pnt-mediated activation(Webber et al., 2018).

In contrast to the VPC system, ERK works only by relievingrepression during the induction of ind in the early fly embryo. Inthis case, ERK phosphorylates Capicua (Cic), an HMG-boxrepressor that acts as a sensor of ERK signaling in Drosophila andother organisms (Jimenez et al., 2012). In the absence of ERKsignaling, Cic is localized predominantly to the nucleus, where itrepresses ind through the highly conserved Cic-binding sites withinthe ind enhancer (Ajuria et al., 2011). One of the mechanismsproposed for the signal-dependent relief of gene repression by Cicinvolves the ERK-dependent nuclear export of Cic, followed by itsdegradation in the cytoplasm (Fig. 4B) (Grimm et al., 2012). Theexpression of ind, however, can be detected before any significantreduction in the nuclear levels of Cic, suggesting that the ERK-dependent relief of gene repression can be achieved while Cic isstill in the nucleus (Lim et al., 2013). Presumably, ERKphosphorylates Cic while it is still bound to its target enhancers.One possibility is that, similar to the disruption of the proteincomplex that represses lin-39 in the VPC system, phosphorylationby ERK causes rapid disruption of Cic interactions with its bindingpartners involved in gene repression. These events would befollowed by dissociation from DNA and slower export from thenucleus. Interestingly, ERK also phosphorylates Groucho (Gro), abroadly expressed co-repressor involved in the regulation of ind aswell as a number of other genes (Hasson et al., 2005; Helman et al.,2011; Cavallo et al., 1998). Gro acts as a non-DNA-binding co-repressor that interacts with other DNA-binding transcriptionfactors, such as Cic, that are crucial for silencing gene expression.Phosphorylation interferes with the co-repressor function of Grobut does not lead to its degradation (Cinnamon et al., 2008). Thephosphorylated form of Gro persists in the nucleus even after the

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nuclear levels of Cic are re-established after termination of ERKsignaling, potentially providing a long-term memory mechanismfor the transcriptional interpretation of the transient ERK signal(Helman et al., 2011). Although it is known that Gro is required forCic repression based on genetic perturbations, there is no directevidence that they form a complex on the regulatory DNA of targetgenes.The nature of the transcriptional response during the ERK-

dependent otx expression in Ciona is more poorly understood thanin the two systems described above. What is known is that ERKphosphorylates the Ciona ETS1/2 factors that act as essentialactivators of otx expression (Farley et al., 2015; Bertrand et al.,2003). It remains to be determined whether these factors areconverted from repressors into activators, such as LIN-1 andLIN-31 in C. elegans, and other ETS factors found in vertebratesand invertebrates or behave completely differently (Maki et al.,2004; Rebay and Rubin, 1995; Sharrocks, 2001). Although weknow that ETS factors are required for otx expression, it is stillnot even clear in the Ciona system whether ERK directlyphosphorylates ETS1/2, or whether it, for example, inactivatestheir repressors.Note that phosphorylation of transcription factors that are already

expressed in the cell is only one of the strategies by which ERK cancontrol its transcriptional targets. Cascade-type mechanisms,whereby a protein product of a gene induced by ERK activationcan function as a new regulator of additional downstream targetgenes, add another level of complexity to transcriptional control.

This mechanism is well documented for the Drosophila ETSfactors, which are expressed upon relief of their repression by Cic,and control multiple genes involved in ERK-dependent cellfunctions (Dissanayake et al., 2011; Jin et al., 2015). In additionto targeting the transcription factors that work at the level ofregulatory DNA, active ERK can also affect gene expression moredirectly, by phosphorylating the components of basal transcriptionmachinery. Recently, it has been shown in human cells that ERKphosphorylates INTS11, a catalytic subunit of an RNA polymerase-associated complex called the integrator (Yue et al., 2017).Interestingly, ERK can also act to modulate the chromatinlandscape at its target genes. For example, in cancerous prostatecells, ERK-mediated phosphorylation of an ETS factor causesdissociation of components of the polycomb repressive complexfrom the chromatin, which then creates a permissive environmentfor transcription (Kedage et al., 2017).

Biochemical characterization of the full repertoire of mechanismsavailable for transcriptional interpretation is essential to completeour understanding of inductive ERK signaling. We can begin byasking how ERK interacts with transcription factors in space andtime, and at different levels of pathway activation. When ERKtargets a transcriptional repressor, what structural changes lead tode-repression (as occurs for Cic) versus conversion to the activatorstate (as occurs for LIN-31)? Does phosphorylation simply switchoff a repressive function, change subcellular localization, involvea co-factor or change the function of the protein altogether?Although dynamic changes in subcellular localization are most

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Fig. 4. ERK-dependent control of transcription factoractivity. (A) Both de-repression and conversion to activationoccurs in C. elegans. In the absence of dpERK, LIN-1 and LIN-31 remain in a repressive complex. Phosphorylation (P) bydpERK relieves LIN-1 of its repressive function, allowingexpression of the target gene (TG) (upper panels). In addition,LIN-31 can be converted to an activator of 1° fate target genessuch as lin-39 (Tan et al., 1998; Wagmaister et al., 2006). Forsome target genes, LIN-1 also becomes an activator (lowerpanels). In this system, induction is analogous to first letting goof the brakes and then pressing on the accelerator of a car.Arrows do not necessarily indicate dissociation of LIN-1 andLIN-31 while in contact with DNA. (B) ind expression inDrosophila relies on de-repression of the transcription factorCapicua (Cic) – similar to relief of LIN-1 repression. Cic binds tothe regulatory region of ind to repress its expression andrequires a co-repressor: Groucho (Gro). ERK activity leads toCic unbinding and export from the nucleus, which then permitsind expression (Lim et al., 2013). In this example, permissiveinduction via de-repression is analogous to letting go of thebrakes on a car without pressing on the accelerator. In thisschematic, the drawing of Gro and Cic does not indicate theformation of a complex since, as of yet, there is no evidence ofphysical interaction.

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readily pursued by live imaging, questions related to changes in theinteraction partners can be addressed using tools such as chromatinimmunoprecipitation (ChIP), which can identify how active ERKinteracts with proteins in the nucleus. For example, ChIP-seqanalysis was recently used to show that ERK-induced activationof the transcription factor Elk1 also leads to histone modificationsthat promote transcription in mouse embryonic fibroblasts(Esnault et al., 2017). Structural analysis will then be essential todescribe how ERK-induced changes between transcription factorsand DNA or other components of transcriptional machinery lead togene expression. In the case of C. elegans VPC induction, theseapproaches could help clarify whether LIN-1 and LIN-31 dissociateon or away from the DNA. The answer may be dependent onwhether LIN-1 is simply de-repressed or whether it also becomesan activator.

What are the origins of robustness?Defects in tissue patterning and morphogenesis can be caused byboth gain- and loss-of-function genetic perturbations of the ERKpathway and its inputs (Runtuwene et al., 2011; Visser et al.,2012; Newbern et al., 2008; Xing et al., 2016; Pucilowska et al.,2012; Vithayathil et al., 2017; Tartaglia et al., 2007; Panditet al., 2007). This conclusion stems from studies of both humangenetic diseases and of developmental processes in modelorganisms (see Box 1) (Jindal et al., 2017). For example, ERKactivation by locally secreted FGF plays a key role in patterningof the mammalian forebrain, a process that can be disrupted byboth loss of ligand and by extending the duration of ligandproduction (Nonomura et al., 2013; Meyers et al., 1998). Theeffects of gain-of-function mutations on ERK activation in vivoappear to be context dependent. Indeed, recent studiesdemonstrate that constitutively active mitogen-activated proteinkinase kinase (MEK) can in fact have divergent effects on ERK

activity in different regions of the embryo (Goyal et al., 2017).Clearly, both lower and upper bounds on the doses, durationsand spatial extents of ERK signals must exist duringdevelopment. How strict are these limits? What are themechanisms that establish them and ensure that they areobeyed during embryogenesis? In all cases studied so far,robust developmental outcomes require coordinated control ofERK signaling at multiple levels, from ligand production totranscriptional interpretation.

Open-loop mechanismsOpen-loop control mechanisms, which by definition do not involvefeedback, are important for ensuring robust signaling outcomes. Foran open-loop control system, the downstream effects of activatedERK do not affect the input signal or change signal transduction.Rather, orthogonal, ERK-independent inputs influence the emergingspatiotemporal patterns of ERK activation by altering signalingtransduction at multiple levels. For example, an open-loop controlmechanism can originate from other signaling systems that disruptprotein interactions in the ERK signaling cascade when activated. Inaddition, expression of the target genes of ERK are often controlledby a number of transcription factors that do not all necessarily interactwith active ERK. ERK-independent transcriptional repressors andactivators therefore offer an open-loop control mechanism at the levelof transcription.

The expression of ind in the early Drosophila embryo is anexample of a patterning event that is robust with respect tosignificant variations in the dose, duration and spatial extent of ERKactivation (Fig. 5). The two-striped pattern of ind, which isestablished by a transient pulse of ERK signaling, persists when theamplitude of this pulse is reduced to a quarter of its value in thewild-type embryo and when the pulse is delayed by almost 1 h (Limet al., 2015; Rogers et al., 2017). Furthermore, the expression of indremains essentially unperturbed when the duration of the ERKsignaling pulse is extended well beyond the normal 1 h timewindowand when it is expanded to the entire blastoderm (Johnson et al.,2017). Part of this impressive robustness can be attributed to the factthat, as discussed above, in this context, ERK works by relievingrepression. As a consequence, the ERK-independent activators caninitiate the expression of ind as long as the strength of the providedERK signal exceeds a threshold value (shown as θ in Fig. 5). Thevalue of this threshold appears to be significantly lower than thesignaling level in the wild-type embryo, which means that theinductive signal can be reduced significantly and still elicit normaltranscriptional response (Lim et al., 2015). Robustness with respectto perturbations in the opposite direction relies on the effects ofERK-independent factors. In particular, the repressors Snail (Sna)and Ventral nervous system defective (Vnd) ensure that ind cannotbe activated in the ventral and ventrolateral part of the embryo,even if ERK activation is expanded beyond its normal domain(Rogers et al., 2017; Stathopoulos and Levine, 2005). Furthermore,diminishing levels of the nuclear localization of the transcriptionalactivator Dorsal limits ind expression on the dorsal side of theembryo. Thus, robust induction of ind relies on several regulatorystrategies, including threshold-dependent responses andcombinatorial effects of ERK-independent activators andrepressors (Samee et al., 2015).

The ERK-independent repressors restraining the wild-typeexpression of ind act at the level of the regulatory DNA.Similarly, in the C. elegans VPC model, the transcriptionalrepressors REF-2 and the MAB-5 repress lin-39 in posterior Pn.pcells to render only a subset of exactly six VPCs, P3.p to P8.p,

Box 1. Developmental abnormalitiesHuman developmental abnormalities have been associated withdisruption of the ERK pathway, in the context of both loss and gain offunction of ERK activity. For example, some individuals on the DiGeorgesyndrome spectrum are haploinsufficient for ERK2 expression, causedby a micro-deletion near the ERK2 locus on chromosome 22 (Newbernet al., 2008). These individuals often exhibit craniofacial and conotruncalabnormalities that stem from disrupted neural crest development.Gain-of-function mutations that occur in many components of the Ras/ERK pathway have been identified in a number of syndromes such asCostello syndrome, Noonan syndrome and cardio-facio-cutaneoussyndrome that are collectively called RASopathies (Tidyman andRauen, 2012). Although mutations in different components of theERK pathway cause distinct syndromes, each is associated withunique sets of developmental abnormalities and many of thephenotypic features of RASopathies overlap. These symptomsinclude craniofacial abnormalities, congenital heart defects,neurocognitive delay and predisposition to certain cancers.

Syndromes that are not associated with mutations in components ofthe ERK pathway, but are linked to deregulated ERK signaling also exist.For example, haploinsufficiencyof nuclear receptor-bindingSET-domainprotein (NSD1) leads to diminished ERK activity in individuals with Sotossyndrome (Visser et al., 2012). This syndrome is characterized by tallstature, craniofacial defects and mental retardation. Loss of function inhuman ribosomal S6 kinase 2 (RSK2) causes Coffin-Lowry syndrome,an X-linked disorder characterized by severe mental retardation inmales (Beck et al., 2015). RSK2 acts as a regulator of ERK signaling,which may impact cell proliferation and differentiation during braindevelopment.

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competent to respond to ERK signals (Alper and Kenyon, 2002).Viewed more broadly, this strategy amounts to prepatterning of acellular response to an extracellular cue and can be implemented inmany ways. For example, during the induction of otx in the Cionaembryo, the effect of FGF is spatially restrained by the Eph/ephrinpathway (another juxtracrine signaling system) that providesnegative control of processes leading to ERK activation and isrequired to restrict otx expression to exactly four cells (Haupaixet al., 2013; Ohta and Satou, 2013). Similar to what happens in theind regulation circuit, the ephrin signals are established by spatiallynonuniform maternal inputs and are independent of the ERKactivation level. This open-loop control strategy appears to beespecially suited for the rapidly developing early stages ofembryogenesis, where inductive signals operate under stricttemporal constraints. Indeed, cell fate patterning in the earlyCiona embryo and ind induction in Drosophila take place on theorder of minutes, within the time scale of a cell cycle (Lim et al.,2015; Nakatani and Nishida, 1994). For patterning processes thatwork on a longer time scale, negative regulators of ERK activationand transcriptional responses can be subordinated to the inductivesignals, leading to a variety of feedback control strategies.

Feedback mechanismsRather than rely on ERK-independent control mechanisms,feedback enables a signaling system to self-regulate, fine-tuningthe spatiotemporal patterns of input signals with their downstreamresponses. Feedback mechanisms have been extensively studiedin the C. elegans VPC model, and contribute significantly to therobust patterning outcomes. One important mechanism is thathigh levels of ERK activity in the VPC nearest the EGF sourceactivate the expression of Notch ligands that signal laterally to theneighboring cells (Chen and Greenwald, 2004; Hoyos et al.,2011; Sternberg, 1988). Notch signaling inhibits ERK activationin those cells by upregulating negative regulators of the ERKpathway, including an ERK phosphatase, lip-1, that counteractsthe effect of ligand-dependent ERK phosphorylation (Bersetet al., 2001; Yoo et al., 2004). The resulting combination of lateralsignaling and negative feedback ensures that the cells adjacent tothe VPC with 1° fate are less sensitive to the EGF input (Zandet al., 2011). Positive-feedback regulation also takes place in theform of ERK-induced EGFR expression in P6.p, which receivesthe most EGF. Moreover, endocytosis-mediated downregulationof Notch receptors in P6.p desensitizes this VPC to lateral Notchsignaling, further amplifying the all-or-nothing response in termsof 1° fate to the locally secreted inductive signal from the AC(Shaye and Greenwald, 2002). In addition to these signalingprocesses, EGF induces migration of VPCs towards the AC torealign displaced cells. The VPC induced with 1° fate migrates up theEGF gradient towards the AC. During this migration, this VPC isexposed to increasing concentrations of EGF that further promote the1° fate. In summary, multiple levels of control confer robustness thatcannot be described by a gradient model alone (Hoyos et al., 2011).These regulatory networks also involve cell cycle control, possiblyrelaxing the time constraint of VPC differentiation (Euling andAmbros, 1996; Miller et al., 1993). The downstream targets of ERKsignaling, LIN-1 and LIN-31, also promote expression of cki-1,which inhibits cell cycle progression during VPC differentiation(Clayton et al., 2008). The same ERK signals involved in cell fateinduction are therefore simultaneously regulating fate specificationand cell cycle progression.

Thus, each of the systems discussed uses several concurrentregulatory strategies to ensure timely and correct responses to

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Fig. 5. Robustness of the ind expression pattern. The spatial and temporalprofiles of ind are remarkably robust to perturbations in ERK input parameters.(A) In the wild-type animal, a pulse of ERK signaling leads to a switch-likeexpression of ind. The wild-type ERK pulse crosses a threshold, θ, at time τ. τalso marks that time at which ind expression turns on in a switch-like manner.(B) (i) When ERK activity is expanded in space, amplitude and duration, theexpression pattern of ind is largely unaffected. Pre-patterned transcriptionfactors should limit the extent of expression even when dpERK expression isexpanded to a larger field of cells. Optogenetic experiments confirm that indexpression is restricted to ventrolateral cells, even when ERK is activethroughout the embryo at maximal levels for an extended period of time(Johnson et al., 2017). (ii) The ind pattern also remains invariant when ERKactivity is only prolonged (Rogers et al., 2017). (C) The ind expression patternis also robust to perturbations that decrease the ERK input. A decreasedduration of ERK activity should not affect ind induction as long as the initial partof the pulse crosses the minimum threshold (θ). If the amplitude of the ERKpulse is decreased, however, θ will be met at a delayed time point (reflectingthe time required for active ERK levels to accumulate). This delay is ∼20 minwhen the ERK pulse is diminished by 25% (Lim et al., 2015).

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inductive signaling by the ERK pathway. Although the jointeffects of the open-loop and feedback mechanisms have beendocumented in multiple developmental contexts (O’Connor et al.,2005; Rogers and Schier, 2011; Rogers et al., 2017; Ribesand Briscoe, 2009), we are still far from assigning the differentialcontributions of multiple mechanisms in any given system.Understanding these differential contributions is needed forestablishing the quantitative constraints that govern ERKsignaling at multiple stages of development, which is in turnessential for probing the origins of the ERK-dependentdevelopmental abnormalities – where ERK signaling has beendisrupted beyond the limits that permit normal development. Suchexperiments will be greatly enabled by new tools that permitexternal manipulation of signaling pathways in vivo as well as livereadouts of pathway activity – as discussed further below.

DiscussionWhen presented with the large and steadily growing number ofpublications about ERK signaling, one may wonder whether wehave already reached saturation and satisfied our curiosity about thishighly conserved signaling system. After all, the majority ofpublications revolve around the same set of components andfrequently pose very similar questions about the specificity,dynamics and robustness of ERK regulation and function. Ourcomparative review of three canonical examples of inductive ERKsignaling argues that although we are still far from answering thesequestions, even in some of the most advanced experimental models,doing so should be an important future goal for the field.Some of the outstanding questions may be addressed by existing

approaches, such as fluorescence correlation spectroscopy formeasuring concentrations and ChIP for studying interactions at thetarget gene loci. At the same time, the field is in crucial need of newtechniques for visualizing and manipulating ERK signaling in vivo.Live reporters of ERK activation have been used quite extensively incultured cells, but their applications in developmental contexts areonly beginning to emerge (Regot et al., 2014). For example, a recentstudy (de la Cova et al., 2017) used signal-dependent changes in thenucleocytoplasmic ratio of an engineered ERK substrate to monitorERK signaling in the VPC system. This study revealed unexpectedoscillatory dynamics of ERK activity in the VPCs. Importantly, thehigher resolution visualization of ERKdynamics showed that differentlevels of the EGF gradient leads to frequency-modulated, rather thanamplitude-modulated, VPC specification. In theory, oscillations inERK activity may be caused by overexpression of an exogenous ERKsubstrate, arising as a consequence of competition between ERKsubstrates and phosphatases (Liu et al., 2011). However, if theseoscillations reflect the endogenous dynamics induced by locallysecreted EGF, we might have to revisit the current models of cell fatespecification in this extensively studied model of inductive ERKsignaling.In addition to live monitoring of ERK activity, new tools to

manipulate ERK signaling inputs in vivo are much needed and arerapidly being developed. Optogenetic systems allow independentcontrol of the spatial extent, dose and duration of signaling withhigh precision in an embryo (Toettcher et al., 2011, 2013). Recently,the optoSOS system, comprising components of the ERK pathwayengineered to respond to light inputs, was shown to strongly activateERK in various contexts during Drosophila embryogenesis(Johnson et al., 2017). This study found that the same, stronglyactivating optogenetic perturbations to the ERK pathway applied atdifferent stages of embryogenesis produced drastically differentphenotypes. Early embryogenesis is highly sensitive to levels of

ERK activity, whereas later stages are more robust (Johnson et al.,2017). This sensitivity remains true for perturbations in space, asectopically activating the ERK pathway in only a few cells in themiddle of the embryo is lethal, whereas overactivation at the poles,where there is the endogenous signal, is not.Much like developmentaldisorders involving hyperactivation of the ERK pathway (Aoki et al.,2013; Runtuwene et al., 2011), the results of studies using thisoptogenetic approach demonstrate that the consequences ofderegulated signaling depend on the developmental context.

Many of the unanswered questions that we highlight withexamples of inductive ERK signaling must be asked for thehandful of other signaling systems that together generate complexityduring development (Housden and Perrimon, 2014). For example,Hedgehog (Hh) signaling in the developing Drosophila wing discand abdominal epidermis relies on filopodial extensions calledcytonemes that carry concentrated Hh signaling components acrossseveral cell diameters (Bischoff et al., 2013; González-Méndez et al.,2017; Chen et al., 2017; Kornberg, 2017). This mode of bringingligands and their cognate receptors together lies in between thelimiting regimes demonstrated by the VPC and otx patterningsystems, respectively. Whereas contact-mediated ERK signaling inthe early Ciona embryo depends on arrangement of cells in a tissue,the Hh signaling contacts are controlled by dynamic cytoskeletalstructures. These cytoplasmic extensions can also be biased to breakthe symmetry of ERK activity in a field of responding cells (Penget al., 2012). Generally, dynamicmorphology of inducing cells, suchas protrusive structures carrying ligand, and of responding cells, asobserved in the VPC system (Grimbert et al., 2016; Huelsz-Princeand van Zon, 2017), may serve as an important control mechanism ofthe absolute numbers of ligand-receptor complexes formed.

Quantitative limits to the signaling parameters of developmentalpathways other than the ERK pathway are also important. Forexample, temporal modulation (through optogenetic techniques) ofthe signals provided by the Nodal pathway can lead to differentpatterning outcomes in the early zebrafish embryo (Sako et al.,2016): different durations of Nodal activity induce qualitativelydistinct responses. In fact, Nodal signaling in the zebrafish acts inconcert with ERK signaling to specify endoderm and mesoderm(Poulain et al., 2006), having opposing effects on activation of thetranscription factor Casanova, which is required for the induction ofa stereotypic pattern of endodermal cells at the zebrafish blastodermmargin (Aoki et al., 2002). This system may be an ideal applicationof dual-input optogenetics to study how the combinatorial actions ofsignaling systems control multiple aspects of tissue patterning andmorphogenesis (Martinez-Arias and Stewart, 2002).

Concluding remarksWe have presented three canonical examples of inductive ERKsignaling in Ciona, Drosophila and C. elegans to demonstrate theimportant unanswered questions related to multiple aspects of ERKdynamics and function. There are a number of issues that need to beresolved to explain how a single pathway, like the ERK pathway,can have such diverse effects during embryogenesis. We need aquantitative understanding of signal initiation, as there may beimportant ligand-receptor dynamics that shape the inputsto signaling pathways. The interpretation of incoming signalsultimately determines the downstream transcriptional responses.In many cases, it is still not known how active ERK interacts withdownstream targets and ultimately alters their functions. Moreover,we must now quantify the context-dependent limits on signalingparameters such as spatial extent, duration and signaling strength tounderstand the origins of the remarkable robustness observed in

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differentiating tissues. Accomplishing these tasks is crucial forlaying down the foundation for a quantitative picture ofdevelopmental ERK signaling and is impossible without well-studied experimental systems, such as those discussed in thisReview.

AcknowledgementsThe authors thank Yogesh Goyal, Granton Jindal, Meera Sundaram, Emma Farleyand Patrick Lemaire for helpful discussions.

Competing interestsThe authors declare no competing or financial interests.

FundingThis work was supported in part by National Institutes of Health grant R01GM086537. Deposited in PMC for release after 12 months.

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