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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
Key
Ras
Raf
MEK
ERK
TF
NucleusTarget genes
Plasma membrane
Growth factor
Receptor tyrosine kinase Kinase
GTPase Transcription factor
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
B Drosophila melanogaster
C Caenorhabditis elegans
P3.p P4.p P5.p P6.p P7.p P8.p
AC
An Vg
An
Vg
a6.5
b6.5
a6.6a6.7
b6.8b6.6
b6.7
a6.8
A6.2A6.4
A6.1A6.3
B6.1B6.2
B6.3 B6.4
a6.5b6.5A6.4
A6.3
B6.2A6.4
A6.2
a6.6 a6.8b6.7
b6.8 a6.7 b6.6
B6.2A6.4A6.3 B6.1
B6.2
B6.4 B6.2
B6.3A6.4 A6.3
A6.2A6.1
A6.4 A6.3A6.1
A6.4A6.3
A P
Cross-section
A P
A Ciona intestinalisA
P
A
P
V
D
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
C Autocrine
B Juxtacrine
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
A
B
ind
LIN-1
TG
ind
LIN-1
Induction
P
P
OFF ON
TG
TG TG
TG
LIN-1 LIN-31
LIN-1 LIN-31
LIN-31
LIN-1 LIN-31
Gro
Cic
P
TGLIN-1 LIN-31
indCic
Gro
P P
Gro Cic
PLIN-31
P
PP
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
A Wild type
Time
Leve
l
dpERK ind�
�
C Decreased
Time
Leve
l
�
��+20
B Increased
Time
Leve
l
(ii) Duration
�
�
Leve
l
Time
(i) Spatial extent, amplitude and duration
�
�
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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