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Submitted version

Resolving time and space constraints during neural crest formation and

delamination

Jean-Loup Duband1, 2, Alwyn Dady1, 2, *, and Vincent Fleury3

1. Université Pierre et Marie Curie-Paris 6, Laboratoire de Biologie du Développement, 75005

Paris, France

2. CNRS, Laboratoire de Biologie du Développement, 75005 Paris, France

3 Laboratoire Matière et Systèmes Complexes, CNRS et Université Denis-Diderot-Paris 7,

75013 Paris France

* Present address: Children's Hospital of Pittsburgh, Rangos Research Building, Pittsburgh, PA

15201, USA

Author for correspondence: Jean-Loup Duband, Laboratoire de Biologie du Développement,

Université Pierre et Marie Curie, 9 quai Saint-Bernard, 75005 Paris, France

email : jean-loup.duband@upmc.fr; tel : 00 33 1 44273645; fax : 00 33 1 44273497

Running title: Time and space during neural crest development

Key words: neural crest, induction, delamination, epithelium-to-mesenchyme transition, migration, sorting, cadherins, core EMT regulatory factors

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Abstract

A striking feature of neural crest development in vertebrates is that all the specification,

delamination, migration, and differentiation steps occur consecutively in distinct areas of the

embryo and at different timings of development. The significance and consequences of this

partition into clearly-separated events is not fully understood yet, but it ought to be related to the

necessity of controling precisely and independently each step, given the wide array of cell types

and tissues issued from the neural crest throughout the embryo and the long duration of their

development spanning almost the entire embryonic life. In this review, using the examples of

early neural crest induction and delamination, we discuss how time and space constraints

influence their development and describe the molecular and cellular responses that are employed

by cells to adapt. In the first example, we analyze how nascent neural crest cells that are initially

mingled with other neurectodermal progenitors after induction in the blastoderm are

progressively segregated from the other neural tube and ectoderm populations and positioned at

the apex of the neural tube prior to migration, owing to the interplay between cadherin-mediated

selective adhesion and global cell movements. In the second example, we examine how

cadherins drive the entire process of neural crest segregation from the rest of the neurectoderm

by their dual role in mediating first cell sorting and cohesion during neural crest specification

and later in promoting their delamination. In the third example, we describe how the expression

and activity of the transcription factors known to drive epithelium-to-mesenchyme (EMT)

transition are regulated timely and spatially by the cellular machinery so that they can

alternatively and successively get involved first in neural crest specification, then in

delamination. In the last example, we briefly tackle the problem of how EMT-inducing factors

may elicit different cell responses in neural crest and neural tube progenitors.

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The term neural crest stems from the fact that, in many species and particularly in some urodele

amphibians, its cellular components protrude over the dorsal midline of the embryo after

delaminating from the closing neural tube (Le Douarin and Kalcheim, 1999; Trainor, 2013); and

as a matter of fact, it is precisely at that time that this cell population individualizes as a distinct

entity, fully segregated from the rest of the neurectoderm from which it derives. This

morphological feature has long served to define the neural crest as a population of migrating

cells that arise from the dorsal neural tube and populate various sites in the embryo, where they

undergo terminal differentiation. However, a variety of cell-tracing studies in chick and Xenopus

clearly established that neural crest cells are not born in the neural epithelium at the time when

they initiate migration but long before, by a mechanism that is separate from that of induction of

the neural plate, the primordium of the central nervous system (Aybar and Mayor, 2002; Basch

et al., 2006; Betancur et al., 2010; Ezin et al., 2009; Milet and Monsoro-Burq, 2012; Prasad et

al., 2012). Thus, despite its intimate physical association and intricate functional connections

with the central nervous system, the neural crest must no longer be regarded as a byproduct of

the neural tissue but as a genuine, distinct embryonic structure which by itself contributed to the

emergence of the vertebrates during evolution and their adaptation to all the ecological niches on

Earth.

In birds, fishes, and amphibians and probably also in mammals, neural crest progenitors

are specified during gastrulation, in the blastoderm, before its subdivision into neural and non-

neural epithelium. As neurulation proceeds, these progenitors are progressively positioned, first

in the elevating neural folds, then in the dorsal aspect of the neural tube. Later on, by a

mechanism related to epithelium-to-mesenchyme transition (EMT), they delaminate from the

neural tube, and owing to a complex code involving multiple guidance cues, they disperse

through restricted pathways. Finally, they colonize many different territories thoughout the body,

where they eventually give rise to a large collection of cell types. Thus, development of the

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neural crest divides into four well-defined steps: the premigration phase, consisting essentially in

the early induction of progenitors and their maturation as a population of cells endowed with the

competence to undergo migration and to give rise to multiple lineages; the delamination phase

which allows their physical separation from the neural tube; the migration phase; and, finally the

ultimate phase of differentiation in the sites of election. Intriguingly, these steps all occur in

distinct areas of the embryo and at different timings during development, the course of neural

crest ontogeny spanning almost the entire duration of embryonic life: specification in the

blastoderm during gastrulation and early neurulation, delamination from the dorsal neural tube

along the embryonic midline at the end of neurulation, migration and differentiation throughout

the course of organogenesis and late embryogenesis in as diverse locations as the vertebral

column, the skin, the aorta, the periphery of the brain, the face, the heart, or the gut. Although

not unique, it is not quite common that during embryogenesis, cells execute sequentially their

main developmental steps in separate locations. Therefore, time and space are both important

parameters that may underlie the entire process of neural crest development and may impinge on

the regulatory processes involved; and there are now many situations in which mismatches

between them may cause severe developmental anomalies. In the present chapter, using selected

examples taken from specification and delamination, mainly in the chick and frog but also in the

zebrafish and mouse, we will address the question of how space is integrated during neural crest

development and how this is regulated over time.

Integrating space during neural crest induction and specification: roles of cell movements

and cadherin-mediated cell sorting

The generation of neural crest progenitors in the blastoderm results from the combined action of

various signals mediated by diffusible morphogens of the Wnt, bone morphogenetic protein

(BMP), and fibroblast growth factor (FGF) families as well as by retinoic acid, emanating from

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the direct environment and acting sequentially or in synergy on a restricted cell population of the

neurectoderm. In response to these signals, cells express a gradually more complex and specific

repertoire of transcription factors assembled into a gene regulatory network, that assigns them

their neural crest identity and allows them to segregate from the ectoderm and neural plate

(Betancur et al., 2010; Groves and LaBonne, 2014; Huang and Saint-Jeannet, 2004; Milet and

Monsoro-Burq, 2012; Prasad et al., 2012; Sauka-Spengler and Bronner-Fraser, 2008).

The first manifestation of neural crest induction during gastrulation is the collective

expression of the transcription factors Pax-7 in chick, Pax-3 in Xenopus, Msx-1/2, and Zic-1.

Expression commences in a territory at the border between the prospective neural plate and

ectoderm, hence its name neural plate border, and which encompasses progenitors of the future

neural crest, as well as those of the dorsal neural tube and ectodermal placodes. Although

individually these factors are neither restricted to the neural plate border nor sufficient to drive

neural crest specification, their combined effect is sufficient to restrict progressively the potential

of cells to adopt ectodermal or neural plate fate, while providing them with the competence to

acquire a neural crest identity. For this reason, these genes have been regrouped under the term

of border specifiers. The second step, named specification, is aimed at consolidating this identity,

in particular to acquire unique adhesive, proliferative, migratory, multipotency and survival traits

that enable neural crest progenitors to undergo EMT and disperse throughout their migratory

routes. This is achieved by the cumulative expression of the neural crest specifier genes,

including members of the Snail and SoxE families of transcription factors, as well as a variety of

other factors such as Foxd-3, Ets-1, AP-2 and Id genes.

Strikingly, the expression patterns of the various border specifiers that have been resolved

in different species reveal that at onset of induction, the neural plate border is not a well-defined

and delineated territory (Basch et al., 2006; Ezin et al., 2009; Khudyakov and Bronner-Fraser,

2009; Milet and Monsoro-Burq, 2012). By combining the techniques of lipophilic dye tracing

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and time-lapse imaging, Ezin et al. (2009) performed fate-mapping studies in chick and were

able to trace precisely the position of neural crest progenitors over time, from early induction to

their final position in the dorsal neural tube. They found that at gastrulation, neural crest

progenitors receiving the induction signal are scattered in the blastoderm in a broad domain

corresponding to the BMP-4-producing region and are mingled with other neurectodermal

progenitors. During neurulation, the neural plate border undergoes spectacular morphological

changes from a short, wide and flat strip of cells with a horse-shoe shape in the anterior

blastoderm into two long, narrow and protruding neural folds that appose and fuse together along

the embryonic midline. During this process, ectodermal, placodal, neural crest and neural plate

progenitors initially dispersed and mixed in the blastoderm become spatially segregated and

patterned into highly-ordered, compact entities along the rostrocaudal and dorsoventral axes.

How is this process achieved? Ezin et al. (2009) revealed that the dynamic displacement of the

neural plate border and sorting of its different cell components occurs as a result of tightly-

coordinated movements of convergence, extension, reorientations as well as cell mixing,

coincidently with the movements of folding, rolling and bending of the neural plate into a hollow

neural tube. These observations therefore uncover the previously-unsuspected importance of cell

movements and rearrangements in the progressive definition of the neural crest territory (and by

the way, that of the other cell populations derived from the neurectoderm) during neurulation and

its confinement to the dorsal neural tube prior to delamination.

Movements of convergence extension cannot, however, explain by themselves the

regroupment of neural crest progenitors and their segregation from the other cells with which

they are mingled. Other cellular events, such as cell cohesion and cell sorting, have then to be

invoked to account for the whole process. Interestingly, recent studies in the zebrafish on the

mechanism of specification of the ventral neural tube progenitors by the inductive signaling

activity of Sonic hedgehog (Shh) (Xiong et al., 2013) provide clues about the sequence of events

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that might be responsible for neural crest regroupment after induction. Indeed, in this species,

instead of being static in a stable epithelium as often assumed in classical models, both the Shh-

producing cells in the axial mesoderm and the Shh-responding cells in the neural tube are motile

and capable of exchanging neighbors. As a result, morphogen signaling across the tissue is

spatially noisy, and neural progenitors being exposed to variable doses of Shh over time and

space exhibit a large range of responses and distribute first in a salt-and-pepper specification

pattern. It is only later that the rearrangement of the different neural progenitors of the neural

tube into sharply-bordered domains is achieved by a cell-sorting mechanism operating in a

gradually-more static population. These observations indicate that specification and positioning

are separate in time and that cell sorting acts to refine spatial patterning by inductive signals. The

fact that the blastoderm is subjected to intense cell movements during induction of the neural

plate border and that emission of the BMP-4 inductive signal occurs in a broad band covering a

large portion of the blastoderm (Ezin et al., 2009) suggests that the same paradigm may apply

during formation of the neural crest.

The question now is by which mechanisms neural crest cells would be sorted from the

other populations of the neurectoderm ? Cell sorting results primarily from the selective

recognition and adhesion of cell populations expressing different repertoires or doses of

molecules of the cadherin family (Halbleib and Nelson, 2006). In the case of neural crest cells in

the chick, until recently the prevailing assumption was that, due to their integration into the

dorsal neural tube, they express primarily N-cadherin, whereas in the overlying ectoderm cells

instead express E-cadherin. In addition, it was generally believed that such a situation is

established precociously during early neural induction and persists throughout neurulation until

delamination (Kalcheim, 2000; Powell et al., 2013; Strobl-Mazzulla and Bronner, 2012a;

Taneyhill and Schiffmacher, 2013; Theveneau and Mayor, 2012). Therefore, cadherins were

essentially regarded as cell-cell adhesion molecules maintaining cell cohesion among neural

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crest cells until EMT. However, recent observations suggest that their role may extend beyond

the sole phase of delamination.

In the chick, our recent data show that there is no mutually-exclusive partition of E- and

N-cadherins in the prospective ectoderm and neural epithelium until they are entirely segregated

at completion of neurulation (Dady et al., 2012). Indeed, in a large anterior half of the embryo,

E-cadherin is at gastrulation uniformly expressed throughout the superficial layer of the

blastoderm while N-cadherin is restricted to the primitive streak, the mesoderm and the

notochord. It is only very progressively that E-cadherin is replaced by N-cadherin in the neural

plate, with a relatively-long time period where both molecules are coexpressed in cells. Thus,

rather than defining precociously the embryonic territories fated to become neural, N-cadherin

upregulation occurs secondarily as a result of neural induction and early neurulation. Moreover,

a detailed survey of the spatiotemporal expression patterns of E- and N-cadherins throughout

neurulation revealed a much more intricate pattern than previously thought (Dady et al., 2012).

At the most anterior levels, from the forebrain to the hindbrain, the E- to N-cadherin switch is

complete at late stages of neurulation even after neural tube closure, while in the trunk, it occurs

earlier during fold elevation. This indicates that this switch is not correlated with the movements

of neurulation, and fully support previous genetic studies in the mouse and zebrafish showing

that in the absence of N-cadherin, onset of formation of the neural tube occurs normally and that

the first signs of malformations are detected relatively late (Hong and Brewster, 2006; Radice et

al., 1997). Which morphogenetic events are then driven by the E- to N-cadherin switch? Studies

in the zebrafish showed that the lack of N-cadherin prevents cells to establish stable protrusive

activity and causes alterations in their radial intercalation in the neural epithelium (Hong and

Brewster, 2006). This suggests that N-cadherin plays a role in cell sorting and in tissue

stabilization, implying that changes in the expression patterns of E- and N-cadherins would

correlate primary with the segregation of neural and ectodermal progenitors.

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Still, neither E-cadherin nor N-cadherin can account for the subdivision of the blastoderm

into three distinct territories (and even four when also considering the placodes), and the

implication of a third partner has to be invoked. Interestingly, the expression pattern of cadherin-

6B in the chick is suggestive of a role for this cadherin in the segregation of the neural crest

contingent from the rest of the neurectoderm and its regroupment at the interface between the

ectoderm and neural tube, as originally proposed by Nakagawa and Takeichi (Nakagawa and

Takeichi, 1995, 1998). Far from being induced late in the dorsal neural tube after neural crest

specification, cadherin-6B appears very early in the neural plate border slightly after induction of

the border genes Pax-7 and Msx-1 and before neural crest specifiers. In addition, consistent with

a role in neural crest cell sorting, it distributes at first as a punctate pattern and becomes

gradually more intense and compact as neural crest progenitors accumulate at the apex of the

neural tube (Dady and Duband, in preparation). Thus, structuration of the blastoderm into

sharply-delineated, contiguous cellular compartments is likely to result from the dynamic

interplay between E-cadherin, N-cadherin and cadherin-6B over time during neurulation. In this

regard, it is worth-mentionning that cadherin-6B has been shown to be upregulated by BMP-4

signaling (Sela-Donenfeld and Kalcheim, 1999), while N-cadherin is in contrast under the

control of FGF and the Sox-2 transcription factor, an early definitive marker for neural tissues

(Linker and Stern, 2004; Matsumata et al., 2005; Uchikawa et al., 2011).

In order to be efficient in a large scale and not limited to a few neighboring cells,

cadherin-mediated cell sorting must be coordinated with dynamic cell movements so that cell

populations can first travel and reorganize over long distances to exchange neighbors and then

become more static and cohesive to stabilize their mutual associations. How are these

movements driven and regulated during neural crest induction and specification? In chick, a

possibility is that they are synchronized with the movements of intercalation occurring nearby

the primitive streak (Voiculescu et al., 2007; Ybot-Gonzalez et al., 2007). However, the limited

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range of these movements along the primitive streak cannot account for the large amplitude of

the so-called polonaise movements thoughout the blastoderm. Biophysical studies based on high

resolution microscopy and fine particle tracking in chick allowed to decipher cell movements at

a large scale over the entire blastoderm, from gastrulation to the end of neurulation, and to define

the physical parameters, such as viscoelasticity and mechanical forces, that drive the shaping of

the embryo (Fleury, 2012). Prior to gastrulation, the blastula exhibits roughly three rings of cells

of different sizes, cells being smaller internally and larger at the periphery. At that time, two

types of movements can be observed, a radial one involving cells at the periphery responsible for

the expansion of the blastoderm over the yolk, and a rotatory one in the center. This creates large

vortices forming a quadrupolar flow. As the embryo initiates gastrulation, propagation of the

mesoderm exerts a traction on the epiblast, stretching and folding the blastoderm

anteroposteriorly. Importantly, folds form at boundaries between cell types and match to the

expression patterns of cadherins in the neurectoderm, segregating the tissue into domains of

different cell sizes and cell-adhesion properties and separated by deep furrows (Fleury,

unpublished). While E-cadherin is progressively replaced by N-cadherin in small cells in the

medial part of the blastoderm, cadherin-6B becomes specifically expressed in the folds. Thus,

folding in the blastoderm produced by differential cell movements may drive the segregation of

the diverse cell populations issued from the neurectoderm.

All together the different analyses described above suggest a model in which, rather than

being de facto a well-defined area of progenitors all sharing the same differentiation potential

that is passively moved toward the embryonic midline by the movements of neurulation, the

neural plate border constitutes an unsteady population that is subjected to intense cellular

reorganizations and is progressively partitioned by the interplay between physical events

operating in the entire blastoderm and biochemical factors acting locally (Figure 1). It would be

interesting to analyze how and to which extent this model developed for a flat embryo like in

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chick must be modified to adapt to spheric embryos like in frog and fish.

Coordinating neural crest cell delamination timely and spatially: regulation of cadherin

activity

By analogy to the now-classical models of EMT developed for cultured cell lines and cancer

cells (Peinado et al., 2007; Thiery et al., 2009; Thiery and Sleeman, 2006), a model has been

proposed for chick neural crest cells in which EMT would occur through repression of cadherins

(namely N-cadherin and cadherin-6B) by a mechanism involving the so-called core EMT

regulatory factors, Snail-1/2, Zeb-2/SIP-1 and Twist-1, and elicited by Wnt-1 and Bmp-4 signals

(Duband, 2010; Kalcheim, 2000; Powell et al., 2013; Strobl-Mazzulla and Bronner, 2012a;

Taneyhill and Schiffmacher, 2013; Theveneau and Mayor, 2012). However, although widely

admitted, this model must be reevaluated at the light of the many progresses accomplished

during the last decade on the comprehension of the intimate molecular processes regulating both

spatially and temporally the sequence of events leading to EMT.

An important point concerns how cadherins are regulated during delamination: what is

the exact timing of their expression and what are their functions in this event? Moreover, a new

question has emerged gradually during the last years: are cadherin-6B and N-cadherin the only

cadherins involved in neural crest EMT in chick? Regarding cadherin-6B, the sharp diminution

of their messages at onset of EMT is indicative of a role in cell delamination (Nakagawa and

Takeichi, 1995; Taneyhill et al., 2007). In addition, Snail-2 has been shown to bind specifically

to E boxes in the regulatory sequence of the cadherin-6B gene and to control directly its

expression (Taneyhill et al., 2007). Finally, functional studies revealed that at cranial levels,

knockdown of cadherin-6B leads to premature neural crest cell emigration, whereas its

overexpression blocks their migration (Coles et al., 2007). Thus, all these data concur with the

idea of transcriptional repression of cadherin-6B being a major triggering event at onset of EMT.

Interestingly, recent studies suggest that the precise timing when EMT occurs is rather defined

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by posttranscriptional regulation of cadherin-6B activity (Fairchild and Gammill, 2013). Indeed,

tetraspanin-18, a member of the tetraspanin family specific of epithelial cells, is abundantly

expressed in premigratory but not migratory cranial neural crest cells. Tetraspanin-18 functions

as a stabilizer of cadherin-6B proteins at the cell surface, and its downregulation under the

control of Foxd-3 is required for neural crest cells to initiate migration. These data illustrate the

complexity of the mechanisms controling the timing of neural crest cell delamination: while

cadherin-6B expression is under the control of Snail-2, tetraspanin-18 acting as a cadherin-6B

stabilizer is itself under the control of Foxd-3.

Several observations, however, argue in favor of alternative or additional roles for

cadherin-6B during delamination. First, its messages persist in the dorsal neural tube after

completion of neural crest cell emigration, indicating that its function may not be restricted to the

temporal control of intercellular adhesion during neural crest EMT (Nakagawa and Takeichi,

1995). In species like mouse and zebrafish, cadherin-6, a close relative to cadherin-6B, is not

downregulated from the surface of neural crest cells after delamination, and at trunk levels in the

chick, cadherin-6B proteins remain present during the early steps of migration (Clay and

Halloran, 2014; Inoue et al., 1997; Park and Gumbiner, 2010). Additionally, functional studies

suggest that, rather than maintaining epithelial stability among neural crest cells, cadherin-6B in

chick and cadherin-6 in fish promote their emigration out of the neural tube (Clay and Halloran,

2014; Park and Gumbiner, 2010). Indeed, trunk neural crest cells expressing cadherin-6B have

been shown to exhibit a general loss of epithelial junctional polarity and gain motile properties

prior to delamination, and cadherin-6B was found to be required for deepithelialization of neural

crest cells. Interestingly, this effect is mediated in chick by non-canonical BMP signaling

involving downstream the BMP type II receptor, the LIM kinase and its major target cofilin, and

in fish by controling Rho GTPase distribution in the cytoplasm. Both signaling pathways

ultimately regulate locally the organization of the actin cytoskeleton and promote polarized

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actomyosin contraction necessary for disruption of apical cell junctions (Clay and Halloran,

2014; Park and Gumbiner, 2010, 2012).

The discrepancy between the data in favor of a role of cadherin-6B in cell-cell adhesion

and those supporting intracellular signaling activity regulating actin dynamics has been

interpreted in terms of differences between cranial and truncal neural crest cells (see e.g.

Taneyhill and Schiffmacher, 2013); and, it is now clearly demonstrated that both populations

differ radically in their modes and kinetics of delamination (Duband, 2010). However, there are

objectively no solid reason for regional differences in cadherin-6B function and regulation, as

many of its partners (catenins, cytoskeletal elements) and regulators (Snail-2, Foxd-3) are found

at both cranial and truncal levels. In addition, a question remains in the signaling model as to

how cell cohesion is maintained among neural crest cells until delamination if the role of

cadherin-6B is solely restricted to disruption of cell-cell contacts. This is not consistent with its

precocious expression during neural crest induction. A possible explanation may reside in the

fact that these experiments differ in an important detail, i.e. the timing when cadherin-6B

expression was affected, and this might be sufficient to account for the differences observed.

Indeed, Coles et al. (2007) modified cadherin-6B expression at cranial levels less than 6 hours

before delamination, while Park and Gumbiner (2010) affected it at trunk levels more than 12

hours before. Because the adhesive and signaling functions described for cadherin-6B are not

mutually exclusive, another scenario can be proposed that would reconcile both models (Figure

2). Both in the head and trunk, cadherin-6B would promote cell sorting during neural crest cell

specification and maintain their cohesion until delamination while activating signaling pathways

that would act as a negative feedback loop and set the grounds for future delamination by driving

polarized changes in the actin cytoskeleton dynamics. Whether both activities may occur

concurrently or sequentially prior to delamination is a critical point that remains however to be

clarified. At cranial levels, additional events such as the stabilizing activity of tetraspanin-18

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may complement this process to coordinate neural crest cell EMT into a massive emigration.

Thus, according to this scenario, premature inhibition of cadherin-6B signaling would block the

cascade of events leading to delamination, while late inhibition of its adhesive function at a time

when polarized contraction of actomyosin has already been activated would in contrast

precipitate emigration. Conversely, early overexpression of cadherin-6B in neural crest cells

would enhance emission of the signals activating cytoskeleton dynamics and therefore promote

delamination, whereas its late overexpression can oppose the diminution of endogenous

cadherins and block delamination. Regardless the precise sequence of events controling its

expression and function, cadherin-6B must therefore be considered as a unique element of the

developmental program responsible for the segregation of the neural crest population from the

rest of the neurectoderm by mediating first their sorting during specification and later by

inducing EMT.

As far as N-cadherin is concerned, it is generally considered as the major cadherin to be

repressed at the time of neural crest cell delamination. In support to this assumption, it has been

demonstrated that at truncal levels in chick, delamination involves cleavage of N-cadherin by the

metalloprotease ADAM-10 under the control of BMP-4. In addition, the released cytoplasmic

fragment of the N-cadherin molecule translocates to the nucleus and stimulates cyclin-D1

transcription and cell delamination (Shoval et al., 2007). This model presents a certain interest in

that, as for cadherin-6B, it is consistent with both the adhesive and signaling activities of

cadherins. However, it has not been validated yet for anterior levels, and given the growing

evidence for the heterogeneity in the modes of neural crest delamination along the embryonic

axis, it may be true only for the lower trunk of the embryo. Moreover, it should be stressed that

the previous analyses on N-cadherin distribution on which this model relies either did not

employ markers for premigratory neural crest cells or were not performed systematically at all

axial levels and at all stages throughout neural crest development (Akitaya and Bronner-Fraser,

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1992; Duband et al., 1988; Hatta et al., 1987; Park and Gumbiner, 2010; Shoval et al., 2007).

Thus, several recent studies argue that N-cadherin may not be implicated at all or at least very

secondarily in neural crest cell delamination at least in the rostral half of the embryo. A detailed

description of the kinetics of neural crest cell delamination at the midbrain level in chick and

mouse (Lee et al., 2013) revealed that a large proportion of the delaminating cells does not

express N-cadherin but rather E-cadherin which is gradually repressed as they separate from the

neurectoderm. Interestingly, these cells are located proximal to the non-neural ectoderm in the

neural folds and correspond to the mesectodermal contingent of the cranial neural crest (i.e. cells

at the origin of bones, cartilage and other non-neural derivatives). Beside these cells, a smaller

contingent situated proximal to the neural part of the neurectoderm expresses both N-cadherin

and E-cadherin prior to migration and provides the neuronal derivatives of the cranial neural

crest. These observations suggest that rather than controling the timing of initiation of cell

delamination, N-cadherin expression would constitute an early neuronal signature in the

premigratory neural crest population. In a systematic analysis of the repertoire of cadherin

expression throughout neural crest formation until their complete segregation from the neural

tube, we found that at least in the head and upper trunk, N-cadherin expression is induced

progressively in the neural part of the neurectoderm during neurulation but not in neural crest

progenitors, including those which adopt a neuronal fate (Dady et al., 2012; Dady and Duband,

in preparation). Significantly, E-cadherin was not confined to the ectoderm but its expression

was found in premigratory cells until delamination or even later, e.g. at the midbrain level. Thus,

N-cadherin repression may not constitute a prominent event during neural crest delamination in

the anterior half of the body. As discussed above, this role is most likely devoted to cadherin-6B

whose repression occurs sharply. The function of E-cadherin in this process is apparently less

critical as its repression proceeds at a slow pace and that, depending on the axial level

considered, it may be expressed on the surface of migrating cells (though at moderate levels) or

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downregulated prior to delamination (Dady et al., 2012; Lee et al., 2013; Dady and Duband, in

preparation). This view is consistent with the expression patterns and functions during

delamination of Snail-2 and Zeb-2, known as bona fide transcriptional repressors of E-cadherin

and cadherin-6B (Peinado et al., 2007; Taneyhill et al., 2007) but not of N-cadherin (Dady et al.,

2012; Dady and Duband, unpublished). Conversely, Sox-2, an activator of N-cadherin

transcription (Uchikawa et al., 2003), is excluded from the neural plate border where it was

found to repress neural crest cell identity (Wakamatsu et al., 2004).

In conclusion, in a new model that differs quite significantly from the previous ones, we

propose that, in chick, cadherins play dual roles during neural crest specification and

delamination and constitute a key element in a coherent program aimed at progressively

segregating these cells from the other neurectodermal populations (ectoderm, neural tube and

placodes), first locally within the neurectodermal epithelium by promoting cell sorting and then

out of it by inducing EMT and migration (Figure 3). An important consequence of this program

is that it restricts spatially and temporally EMT and migration only to neural crest cells without

interfering with the stability and development of the neighboring neural and ectodermal

epithelia. This might explain why EMT can be induced in those tissues only if a neural crest

identity is forced (Barembaum et al., 2000; Cheung et al., 2005; Park et al., 2012; Théveneau et

al., 2007). It should be noted, however, that this model may not apply to Xenopus as in this

species, cadherin-6 is not found in premigratory neural crest (David and Wedlich, 2000) and that

the E- to N-cadherin switch occurs in the neural plate very early at onset of neurulation, with no

transition during which both molecules are coexpressed (Nandadasa et al., 2009). In this species,

which cadherin is recruited to mediate neural crest sorting from the rest of the neurectoderm and

how it is regulated during delamination are not known. The continuous presence of N-cadherin

over the surface of cranial neural crest cells during migration (Theveneau and Mayor, 2012)

raises the intriguing question as to whether onset of migration might not involve a true EMT.

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Coordinating neural crest cell specification and delamination: spatiotemporal control of

the core EMT regulatory factors

Although the moment when neural crest cells exit the neural tube extends generally over less

than an hour (Ahlstrom and Erickson, 2009; Clay and Halloran, 2010), Snail-1/2, Zeb-2 and in a

lesser extent, Twist-1 all appear in neural crest progenitors early during specification long before

onset of EMT. In addition, as discussed above, their major cadherin targets undergo different

temporal patterns of repression. This situation contrasts strikingly with that found in the

primitive streak during gastrulation, where ingressing mesodermal cells express Snail-2 in an

dynamic fashion and immediately execute a complete EMT program with breakdown of the

basement membrane, loss of cell polarity and a rapid E- to N-cadherin switch (Acloque et al.,

2011; Dady et al., 2012; Nakaya et al., 2008). This suggests that in the neural crest, the

transcription-repressing activity of the core EMT regulatory factors is finely tuned over time and

that prior to EMT, these factors may exert additional functions independently of their role in

delamination by regulating different sets of "early" and "late" genes. In this respect, based on

studies in Xenopus, it has been proposed for long that Snail-1/2 plays a role in both specification

and delamination (LaBonne and Bronner-Fraser, 2000; Mancilla and Mayor, 1996), albeit no

specific Snail-1/2 target genes during specification have been identified yet. Therefore, knowing

how Snail, Zeb and Twist expressions and activities are regulated may provide insight into their

missions throughout neural crest development.

EMT is a strictly-controled process driven by multiple, closely-interconnected regulatory

networks centered around Snail, Zeb and Twist and acting at different levels; and in the past few

years, a plethora of factors and pathways modulating their expression, cellular localization,

stability and activity have been identified particularly in the case of Snail-1/2 (Figure 4 and De

Craene and Berx, 2013, for review). The possible implication of a number of them during neural

18

crest cell delamination has been investigated during the last decade, most often at cranial levels

in the Xenopus model, and a temporal sequence of their activity progressively emerges that

might account for the time course of cell specification and delamination.

Transcriptional and translational control of the expression of the core EMT regulatory factors

Transcriptional control of expression of the core EMT regulatory factors is the primary level of

regulation of the EMT program, and this has been particularly investigated for neural crest cells

owing to the knowledge accumulated on the gene regulatory networks supporting their

development (Betancur et al., 2010 and Prasad et al., 2012). As in cancer cells (Lim and Thiery,

2012; Thiery and Sleeman, 2006), EMT being triggered in crest cells by the BMP-4 and Wnt

signaling pathways, the classical transcription factors recruited by these pathways (e.g. Smads

for BMPs and Lef/Tcf for Wnts) have been shown to upregulate directly expression of Snail-1/2

in these cells (Sakai et al., 2005). However, aside from these factors, additional transcription

factors have been identified as potent inducers of core EMT regulators during neural crest cell

specification and delamination. Sox-9, for example, is essential for Snail-2 expression and its

effect is mediated through direct binding to the Snail-2 promoter in synergy with Snail-2 itself,

able to activate its own expression (Sakai et al., 2006). Other factors such as the border

specifiers, Pax-3/7 and Msx-1, can also upregulate Snail-2, but whether it is directly or via other

factors has not been investigated yet. Recently, functional experiments have placed Elk-3, a

member of the Ets family of transcription factors present in neural crest cells early during

specification and migration, downstream of border specifiers and upstream of neural crest

specifiers, including Snail-2, in the genetic cascade accompanying their development (Rogers et

al., 2013). How Elk-3, known to function as a transcriptional repressor, control these genes is not

known. On the other hand, c-Myb, a transcription factor with well-known functions in

hematopoiesis but also expressed in premigratory and early migrating neural crest cells, has been

19

found to control expression of Snail-2 and Twist-1 (Betancur et al., 2014; Karafiat et al., 2005).

Whether this cohort of factors is responsible for modulating expression of Snail-1/2 (as well as

Twist-1 and Zeb-2) between specification and delamination has not been formally established,

and to our knowledge, no transcription factors whose expression correlates with initiation of

delamination have been identified so far. The only described exception is Ets-1, the prototypic

member of the Ets family, that is induced just prior to EMT at cranial levels. Functional studies,

however, indicate that Ets-1 acts merely as a coordinator of delamination for cranial neural crest

cells and not as a direct modulator of Snail-2 expression (Théveneau et al., 2007).

Relatively little is known about the transcriptional repressors of Snail, Twist and Zeb. At

variance with what was found in neural crest cells, Snail-1 has also been reported to bind to an E

box in its own promoter and repress its transcription (Peiro et al., 2006). On the other hand, a

recent study has identified Sox-3 as a potent negative regulator of Snail-2 expression during

chick gastrulation. Sox-3 expressed in the neural plate represses Snail-2 gene, thus protecting

them from ingressing, while in the primitive streak Snail-2 conversely represses Sox-3, activating

EMT and ingression of precursors of the mesoderm (Acloque et al., 2011). Whether this

antagonistic relationship also applies to Sox-2/3 and Snail-2 in neural crest cells is not known.

Although highly plausible, because Sox-2, Sox-3 and Snail-2 are mutally exclusive in neural crest

progenitors and in the neural tube (Uchikawa et al., 2011; Wakamatsu et al., 2004), it remains to

be demonstrated. Indeed, Sakai et al. (2006) reported that Sox-2 does not affect Snail-2 promoter

activity in fibroblast cell lines.

Small non-coding RNAs also emerged as potent modulators of the expression of the core

EMT regulatory factors. MicroRNAs (miRNAs) typically suppress gene expression by

interacting with the 3’-untranslated region of target messengers to repress their translation or

degrade them. A large number of miRNAs have been involved in the control of EMT and,

among them, miR-34 and the miR-200 family are strongly associated with the epithelial state and

20

are downregulated upon EMT (De Craene and Berx, 2013, for a review). While miR-34

represses Snail-1 expression, miR-200s act as silencers for Zeb-1/2. Interestingly, both miRNAs

contain E boxes in their promoters and are directly regulated by Snail and Zeb, thereby revealing

intricate reciprocal feedback loops between miRNAs and their targets (Burk et al., 2008;

Siemens et al., 2011). These miRNAs are, on the other hand, under the positive control of p53

(Chang et al., 2011), an important player of neural crest development (see below). Contrary to

miR-200s and miR-34, miR-9, which is upregulated in breast cancer cells and activated by Myc,

has been found to target directly E-cadherin messages, leading to increased cell motility and

invasiveness in epithelial cell lines (Ma et al., 2010). At the present time, the presence of these

various miRNAs during specification and delamination of neural crest cells has not been

reported. Yet, miRNAs undubiously play an important role in these cells (Mayanil, 2013; Strobl-

Mazzulla et al., 2012). Indeed, in mouse, specific deletion in neural crest of DICER, the RNase

III enzyme required for cleavage of precursor miRNAs into mature miRNAs, leads to

craniofacial and cardiac anomalies (Zehir et al., 2010). Likewise, in Xenopus, loss of DICER or

of FMR1 (fragile-X mental retardation syndrome-1) and FXR1 (fragile-X related-1), two RNA-

binding proteins interacting with the miRNA-induced silencing complex RISC, also causes

craniofacial defects due to strong reduction of neural crest migration (Gessert et al., 2010). On

the other hand, consistent with its role in repressing Zeb expression and preventing EMT,

depletion of miR-200b affects development of cranial neural crest-derived structures but,

unexpectedly, this effect was not found to result from the block in induction and migration.

Depletion of another miRNA miR-96, in contrast, provoke alterations of both induction and

migration (Gessert et al., 2010). Clearly, more systematic analyses of the repertoire, function and

interplay of miRNAs regulating expression of the core EMT factors during neural crest cell

delamination are needed to define precisely how they contribute to its spatiotemporal control.

21

Epigenetic control of the expression of the core EMT regulatory factors

Control of Snail, Zeb and Twist expression may also be part of more general cellular programs

regulated at the epigenetic level, allowing genome-wide coordinated modulation of gene activity.

Though still preliminary, recent studies have highlighted the major transcriptional reprograming

events that accompany neural crest cell specification and delamination and provide insights into

the synergistic control of neural crest specifiers by the epigenetic machinery (Mayanil, 2013;

Strobl-Mazzulla et al., 2012).

DNA methylation by DNA-methyltransferases (DNMTs) is one of the epigenetic

modifications resulting in transcriptional repression of genes. Only DNMT-3A/3B, the DNMTs

responsible for de novo methylations, have been studied during neural crest development.

DNMT-3B is expressed in premigratory and migrating cells but is not restricted to them, and

genetic studies in mouse demonstrated that it is largely dispensable in cranial and cardiac neural

crest cells for migration and differentiation, but is however required in their host tissues,

branchial arches and heart, during the late steps of their development (Jacques-Fricke et al.,

2012). DNMT-3A, in contrast, shows a more-restricted expression pattern than DNMT-3B as it

is at first present in the neural plate border and becomes later confined to premigratory and

migrating neural crest cells. Knockdown of DNMT-3A provokes a severe reduction in

expression of Snail-2 and of a variety of other neural crest specifiers, accompanied by a

remarkable increase in Sox-2/3. Importantly, the latter two genes appeared as direct targets of

DNMT-3A in neural crest cells. These data therefore indicate that DNA methylation acts as a

major molecular switch to turn off neural tube transcription factors in neural plate border cells

and promote neural crest cell fate (Hu et al., 2012). The mechanism by which DNMT-3A is

recruited to the promoters of its target genes in neural crest cells is currently unknown, but it has

been shown to interact with a multiplicity of transcription factors, including some that are highly

relevant to neural crest formation, i. e. p53, Ets-1 and AP-2 (Hervouet et al., 2009). It would be

22

then of interest to establish the complete list of targets of DNMT-3A in neural crest cells and to

determine by which factors it is recruited to them.

Beside DNA methylation, histone methylation and acetylation are the major chemical

modifications which influence chromatin structure and regulate gene expression (Kouzarides,

2007). Methylation of histone H3 at specific amino-acid residues recruits a variety of modifiers

of chromatin and transcriptional activators or repressors, resulting in differential effects on gene

expression. Roughly, trimethylated lysine 4 of histone H3 (H3K4me3) and H3K36me3 are

associated with active transcription, while H3K27me3 and H3K9me3 are repressive marks.

Histone methylation are regulated by the interplay between methyltransferases and

demethylases, notably the Jumonji proteins (Jmj). To date, only Jmj-D2A has been reportedly

implicated in the control of Snail-2 expression during neural crest development (Strobl-Mazzulla

et al., 2010). It is expressed initially throughout the neural plate and at the border and gradually

resolves to the premigratory neural crest. Of interest, its expression is highest during early

specification and then declines until onset of migration at which stage it is almost no longer

expressed. Consistently, loss of Jmj-D2A expression causes dramatic downregulation of neural

crest specifier genes, notably Snail-2, Foxd-3, and Sox-10, but is ineffective on border genes.

Additionally, the location and abundance of the H3K9me3 and H3K36me3 epigenetic marks, as

revealed by chromatin immunoprecipitation, show dynamic occupancy of sites in proximity to

the transcriptional start site of Snail-2 and Sox-10 genes and clearly reflect their transcriptional

state. At initiation of specification, Sox-10 exhibits essentially H3K9me3 repression marks,

while Snail-2 shows a more mitigated pattern with some H3K9me3 repression but H3K36m3

activation marks as well, reflecting an ongoing transcriptional shift from repressed to active.

Later, by the time of migration, the occupancy of the repressive mark H3K9me3 near the Sox-10

and Snail-2 genes is clearly reduced, consistent with high expression of these genes by this stage,

while H3K36me3 marks are unchanged. Correlating with these changes, direct interactions

23

between Jmj-D2A and the regulatory regions of Sox-10 and Snail-2 genes can be detected at

early specification stages but not later during migration. Finally, demethylation��� of H3K9me3 on

the Sox-10 promoter is inhibited upon knockdown of Jmj-D2A in early neural crest cells. These

results therefore indicate that the relief of transcriptional repression by the Jmj-D2A-mediated

demethylation of H3K9me3 marks plays an important role in induction of neural crest specifier

genes.

Other chromatin remodellers, such as CHD-7, a chromodomain helicase DNA-binding

protein homologous to the Drosophila trithorax protein Kismet, have also been shown to control

expression of core EMT regulators during neural crest development (Bajpai et al., 2010). In

Xenopus, CHD-7 is expressed in the neural ectoderm and in premigratory and migrating neural

crest cells. Depletion of CHD-7 specifically targets neural crest specifiers, such as Snail-2, Sox-

9, and Twist-1 and not border specifiers, and it causes major craniofacial defects. In addition,

CHD-7 associates in the nucleus with the PBAF (polybromo- and BRG1-associated factor)

complex, and both remodellers bind to a neural crest-specific distal Sox-9 enhancer and a

conserved genomic element located upstream of the Twist-1 gene. Consistently, CHD-7 and

PBAF-bound regions were preferentially enriched for H3K4me1, a mark previously associated

with enhancers. These data therefore indicate that, complementing Jmj-D2A activity, CHD-7 and

PBAF cooperate to promote expression of neural crest specifiers.

Control of the stability and intracellular location of the core EMT regulatory factors

Snail proteins are by nature highly unstable, including in neural crest cells (Vernon and

LaBonne, 2006). Snail turnover in the cytoplasm is tightly regulated by regulatory mechanisms

involving posttranslational phosphorylation and ubiquitination (De Craene and Berx, 2013).

Schematically, these regulatory mechanisms can be classified into glycogen synthase kinase-3β

(GSK-3β)-dependent and independent processes (Figure 4). In the GSK-3β-dependent process,

24

cytoplasmic Snail-1 proteins are phosphorylated at serine-threonine residues first by casein

kinase-1 then by GSK-3β and they are subsequently processed for ubiquitination by the E3

ubiquitin ligase β-TRCP-1 for degradation (Vinas-Castells et al., 2010). Thus, signaling events

elicited by Wnt and other growth factors causing inactivation of GSK-3β may repress Snail-1

degradation and favor transition toward a mesenchymal phenotype. Given that Wnt-1 signals are

activated in neural crest progenitors prior to delamination, it is tempting to suggest that their

activity may recruit β-TRCP-1 resulting in increased stability of Snail proteins. Interestingly, at

least in chick and mouse, Wnt-1 expression is induced late during neural crest cell specification,

shortly prior to onset of migration (Burstyn-Cohen et al., 2004), suggesting that Snail

stabilization upon inhibition of GSK-3β activity may constitute a late triggering event of neural

crest cell delamination. This hypothesis awaits to be demonstrated, however.

Beside β-TRCP-1, GSK-3β-independent ubiquitin ligases, such as MDM-2 and FBXL-

14, and the F-box protein partner of paired (PPA) have also been reported to target Snail proteins

for degradation (Vinas-Castells, 2010; Lander et al., 2011), and at least two of them, MDM-2

and PPA have been implicated in neural crest development (Daujat et al., 2001; Lander et al.,

2011; Vernon and LaBonne, 2006). Indeed, it has been found in Xenopus that, contrary to Snail-

1, Snail-2 does not contain in its sequence the β-TRCP-1 destruction motif, and inhibiting GSK-

3β causes only marginal increase in Snail-1/2 stability, suggesting that in this species the Wnt-

1/GSK-3β/β-TRCP-1 axis plays only a minor contribution to Snail regulation (Lander et al.,

2011; Vernon and LaBonne, 2006). In contrast, PPA does not target only Snail-1 but also Snail-

2, Twist-1 and Zeb-2, making this pathway a major candidate for regulating the timing of neural

crest cell delamination (Lander et al., 2011; Vernon and LaBonne, 2006). PPA is induced

specifically in premigratory neural crest cells just prior to EMT. In addition, morpholino-

depletion of PPA stabilizes Snail-2 protein, whereas its misexpression promotes its turnover and

inhibits the formation of neural crest precursors. Importantly, Sox-9 and Foxd-3 have been found

25

to repress expression of PPA and oppose its Snail-protein degradation activity when

overexpressed in the neural tube. Thus, prior to EMT, Snail protein levels (and probably those of

Zeb-2 and Twist-1) would be maintained constant through repression of PPA by the combined

activities of Sox-9 and Foxd-3. Then, upon EMT, PPA levels would increase, resulting in higher

Snail-1/2, Zeb-2 and Twist-1 degradation. Consistently, PPA inhibition has been found to cause

migration defects, indicating that high levels of Snail-2 might be deleterious for efficient neural

crest migration (Vernon and Labonne, 2006). Given that neither Sox-9 nor Foxd-3 are

downregulated at onset of migration, which factors relieve PPA repression remains to be found.

Insights into the role of MDM-2 in neural crest cells come essentially from studies on the

regulation of the tumor suppressor p53. Indeed, p53 is one of the main targets of the MDM-2

ubiquitination activity, an important step in the oncogenic process. MDM-2 expression has been

found in murine and avian cranial neural crest cells prior to delamination and to increase in

migrating cells (Daujat et al., 2001; Rinon et al., 2011). Conversely, p53 is at a basal level in

premigratory neural crest and declines after delamination (Rinon et al., 2011). Functional

experiments in chick reveal that stabilization of the endogenous p53 protein by an inhibitor of

MDM-2 activity reduces Snail-2 expression and cell proliferation and inhibits neural crest cell

delamination but not their specification (Rinon et al., 2011). Conversely, loss of p53 resulted in a

marked increase in cranial neural crest progenitors in the neural tube and increased cell

proliferation. Nevertheless, neural crest cells fail to leave the neural tube, suggesting that p53

levels must be finely tuned by MDM-2 activity in neural crest progenitors for delamination and

migration to occur. Further studies by Wang et al., 2011 showed that Pax-3 plays a role in neural

crest cell development, not through its DNA-binding and transcription-regulation activities, but

essentially by blocking p53 function. Pax-3 has no effect on p53 mRNA levels or the rate of p53

synthesis but it reduces p53 protein stability by interacting physically with p53 and MDM2.

These data suggest that during delamination, the Pax-3/ MDM-2/p53 signaling cascade is critical

26

for controling the rate of cell growth and division in neural crest cells. Whether MDM-2

ubiquitination of Snail-2 is required for this process has not be investigated, however. Moreover,

the finding that p53 may control cell invasion in cancer cells by inducing the MDM-2 –mediated

degradation of Snail-2 (Wang et al., 2009) is indicative of complex feedback loops between p53,

MDM-2 and Snail-2 during EMT.

As nuclear Snail proteins degrade more slowly than cytoplasmic Snail, there are

mechanisms that modulate their nuclear trafficking (Figure 4). Thus, two kinases, PAK-1 and

LATS-2, favor nuclear retention of Snail-1, thereby enhancing its stability and activity (Yang et

al., 2005; Zhang et al., 2012). On the other hand, the zinc transporter LIV-1, a STAT-3 target,

has been found to drive nuclear import of Snail-1 in the zebrafish gastrula and promote EMT

(Yamashita et al., 2004), whereas the protein kinase D1 (PRKD-1) by enhancing its nuclear

export in contrast restricts EMT (Du et al., 2010). The roles of LATS-2, LIV-1 and PRKD-1 in

neural crest cell delamination have not been investigated yet, but a recent report indicates that in

Xenopus, PAK-1 expression coincides temporally with their migration (Bisson et al., 2012),

suggesting that it may operate even after delamination. Consistent with this restricted pattern,

expression of a dominant-negative form of PAK-1 blocks migration but does not affect

specification. Intriguingly, PAK-1 is able to phosphorylate Snail-1 and Twist-1 but not Snail-2,

indicating that during migration requirement for Snail-1 and Snail-2 activities are different.

Recently, a novel level of regulation of Snail-1/2 activity has been uncovered in Xenopus,

implicating Twist-1. Although it is commonly presented as an important player in neural crest

cell specification and delamination (Betancur et al., 2010; Milet and Monsoro-Burq, 2012;

Prasad et al., 2012; Sauka-Spengler and Bronner-Fraser, 2008), Twist-1 function has long

remained elusive, and it is only very recently that it has been reevaluated. Unlike Snail-1/2 and

Zeb-2, Twist-1 appears relatively late during the process of neural crest specification and it is

restricted to cranial regions. However, elegant studies by Lander et al. (2013) showed that Twist-

27

1 misexpression or depletion impacts on the levels of Snail-1/2 messages as well as on other

neural crest specifiers. Interestingly, this is not merely through regulation of gene expression but

primarily through direct binding with Snail-1/2 proteins. The C-terminal WR domain of Twist-1

interacts with the N-terminus of Snail via a cluster of GSK-3β-phoshorylated aminoacids; of

note, this interaction does not interfere with the ability of Twist-1 to bind DNA (Figure 5).

Moreover, Snail-1/2 binding stabilizes Twist-1 by competing with PPA association, suggesting

that Snail-1/2 impacts positively on Twist-1 activity. Surprisingly enough, GSK-3β-

phosphorylation of Twist-1 titers Snail-1/2 and diminishes their recruitment to E boxes in the

promoters of their target genes. Thus, although Snail-2 is not directly targeted to degradation by

GSK-3β-phosphorylation (see above), these findings ascribe to GSK-3β a critical role in the

control of its activity via Twist-1, and in the future, determining where and when Twist-1 is

phosphorylated by GSK-3β will be of importance to decipher the mechanisms triggering neural

crest cell EMT.

Finally, recent preliminary data on paladin, an antiphosphatase, may provide interesting

information regarding how the activity of the core EMT regulators can be modulated over time

in premigratory neural crest cells (Roffers-Agarwal et al., 2012). Antiphosphatases have been

shown to bind phosphorylated residues on target proteins and protect them from

dephosphorylation. In chick, paladin is expressed during neural crest development both at cranial

and truncal levels. Decreasing paladin expression precociously during neural crest induction

using morpholinos results in a substantial but not complete reduction in Snail-2 expression at

premigratory stages. Interestingly, only Snail-2 is strongly repressed, the other targets tested

being affected either moderately (e.g. Sox-10) or not at all (Foxd-3, cadherin-6B, and Rho-B).

This indicates that paladin may regulate only a limited set of targets during neural crest

specification. Moreover, cadherin-6B is apparently down-regulated on time in the absence of

paladin, suggesting that essentially Snail-2 activities relevant to specification were altered. At the

28

light of the data presented above, it is tempting to speculate that control of the level of

phosphorylation of Snail-2 by paladin is critical for some of its activities and may vary over time

during specification and delamination. Further experiments, however, are needed to verify this

statement and to better define paladin role and targets. For example, determining which

aminoacids on Snail are targeted by paladin and whether Snail stability and trafficking are

affected upon manipulation of its expression should help better understanding how maintenance

of phosphorylation involving paladin contribute to control of neural crest cell EMT.

Control of the activity of the core EMT regulatory factors

One of the reasons for the ability of the Snail, Zeb and Twist transcription factors to induce

massive phenotypic changes during EMT resides in part in their close interaction with epigenetic

modifiers, allowing genome-wide changes in gene expression. Indeed, their transcriptional

regulatory activity requires the participation of various interacting proteins which results in a

strong transcriptional control of EMT, and this has been particularly well studied in the case of

the E-cadherin gene. For example, DNA methylation of the E-cadherin promoter responsible for

its silencing has been correlated with EMT in cancer cells (Lombaerts et al., 2006) and

associated with increased Snail-1 expression (Cheng et al., 2001). More intriguingly though, it

has also been reported that DNMT-1 can repress E-cadherin expression in the absence of

noticeable changes in DNA methylation patterns in its promoter and that this effect is mediated

by direct interaction with Snail-1 (Espada et al., 2011). During neural crest development,

although DNA methylation by DNMT-3A has been shown to act as a major molecular switch to

turn off neural tube transcription factors in neural plate border cells and promote neural crest cell

fate (Hu et al., 2012), it is not known yet whether Snail-1/2 and the other core EMT regulators

can associate directly or via adaptors with DNMTs to target specific genes.

Beside DNMTs, Snail-1 has been found to associate with a variety of histone modifiers to

29

regulate gene expression (Figure 5). In particular, it induces histone deacetylation of the E-

cadherin gene through the recruitment of Sin-3A in association with histone deacetylases

(HDAC-1/2) (Peinado et al., 2004). Interestingly, recent studies by Strobl-Mazzulla and Bronner

(2012b) uncovered the mechanism by which Snail-2 recruits HDAC to repress cadherin-6B

expression in neural crest cells in chick. They identified PHD-12, an adaptor protein, whose

expression is markedly increased in cranial crest cells just before EMT. PHD-12 loss of function

phenocopies Snail-2 knockdown, inhibiting transcriptional repression of cadherin-6B and

preventing neural crest emigration. PHD-12 and Snail-2 do not bind to each other, but both

directly interact with Sin-3A, which in turn complexes with HDAC. PHD-12 is recruited to the

cadherin-6B promoter during neural crest EMT. Consistent with this, lysines on histone H3 at

the cadherin-6B promoter are hyperacetylated before neural crest emigration, correlating with

active transcription, but deacetylated during EMT, reflecting a shift to a repressive state. Finally,

knockdown of either PHD-12 or Snail-2 prevents deacetylation of the cadherin-6B promoter.

Collectively, these results suggest that repression of the cadherin-6B gene occurs through

binding of PHD-12 and Snail-2 to their transcription start site and E boxes. This allows Sin-3A

and HDAC to be recruited and to deacetylate histone H3 at the promoters, resulting in repression

of transcription. Of interest, the authors mention that this model also applies to E-cadherin

repression. In an other study, Murko et al. (2013) showed that treatment of early embryos using a

pharmacological blocker of HDAC causes neural tube defects at trunk levels, increase in

cadherin-6B expression and a premature loss of epithelial features among neural epithelial cells.

Among the different adaptors with which Snail proteins interact in neural crest cells to

recruit HDAC and drive transcriptional repression of genes are the LIM proteins, characterized

by the presence of two LIM domains in tandem (Figure 5). The first LIM proteins to be

identified were the Ajuba family which were found to function as a corepressor of Snail-1/2

(Langer et al., 2008). Ajuba interacts predominately with the SNAG domain of Snail-1 in cells

30

and accumulates in the nucleus in a SNAG-dependent manner. This interaction with Snail-1

potentiates Snail-1 binding to the E boxes of the E-cadherin promoter and promotes its

repressing activity. Interestingly, a recent study by Ochoa et al. (2012) revealed that contrary to

Snail-1, Snail-2 binds to Ajuba via its Zn-finger motifs. Expression of Ajuba in Xenopus

embryos enhances neural crest development in a Snail-1/2-dependent manner, while conversely,

it depletion phenocopies depletion of Snail-1/2. This study therefore assigns to Ajuba LIM

proteins a critical role in neural crest cell development as Snail-1/2 corepressors. However, their

presence in these cells has not been assessed in this study. Interestingly, Ajuba LIM proteins are

also components of adherens junctions and contribute to their assembly or stability (Srichai et

al., 2004). Their functional interaction with Snail proteins in the nucleus suggests that they are

also important regulators of the dynamics of epithelial cells, linking surface events with nuclear

responses. It would then be informative to determine when and where Ajuba LIM proteins are

expressed in neural crest progenitors to better evaluate their implication in the spatiotemporal

control of Snail-1/2 activity.

More recently, studies in Xenopus and chick identified the LIM domain only protein 4

(LMO-4) as another LIM protein involved in neural crest development (Ferronha et al., 2013;

Ochoa et al., 2012). In both species, LMO-4 is distributed in the neural crest and neural plate

progenitors during early specification to become restricted to neural crest cells at the time of

their delamination and early migration. In addition, functional studies revealed that LMO-4 is a

Snail-1/2-interacting protein that is essential for neural crest development. Morpholino-mediated

knockdown of LMO-4 leads in Xenopus to a deficit in the production of neural crest progenitors

and in chick to a severe reduction in their delamination. Additionally, while misexpression of

LMO-4 in the trunk neural tube in chick is insufficient to induce expression of neural crest

specifiers but speeds up neural crest delamination, in Xenopus, excess LMO-4 leads to ectopic

expression of Snail-1/2, and to a reduction in the expression of Foxd-3, Sox-8/9/10 and Twist-1.

31

These differences between chick and frog are likely not to result from species specificities but

rather to differences in the timing when LMO-4 was overexpressed (late during neural crest

development in chick and early during specification in frog), thereby revealing different

requirements for LMO-4 throughout neural crest development. Detailed analyses of LMO-4

binding specificities further showed that it binds directly to Snail-1/2, but not to other neural

crest specifiers, and this interaction occurs via the SNAG domain of Snail, like Ajuba LIM

proteins (Figure 5). Intriguingly, Snail-1/2 binding to LMO-4 does not lead to HDAC

recruitment, thus raising concerns about its mode of action. The fact that LMO-4 competes with

HDAC for Ajuba binding suggests that it may play a key modulatory role of Snail-1/2

interactions with its partners, thereby selectively targeting distinct sets of genes. It will be

essential then to determine whether LMO-4 recruits specific proteins to Snail-regulatory

complexes on target promoters and to define how Ajuba and LMO-4 are coordinated over time

during neural crest development.

Twist-1 has also been found to recruit epigenetic modifiers as partners for regulating E-

cadherin gene expression. Notably, it has been found to associate with Bmi-1, a member of the

polycomb repressive complex, to suppress E-cadherin transcription through binding to the E

boxes situated in its promoter. Interestingly, Bmi-1 is itself under the direct transcriptional

control of Twist-1 (Yang et al., 2010). Knowing the complex relationships between Twist-1 and

Snail-1/2 in neural crest cells (see above), determining whether Bmi-1 is expressed during their

delamination and may contribute to the control of cadherin expression will be valuable to

decipher these interactions in greater details.

Although a coherent, complete picture of the regulatory networks controling expression

and activity of the core EMT regulators Snail-1/2, Zeb-2 and Twist-1 is still lacking (in part

because studies were performed in different animal models characterized by distinct gene

regulatory networks), two major conclusions can be drawn for these studies (Figure 6). First, a

32

multiplicity of transcription factors and chromatin modifiers and remodellers bind to different

domains in the regulatory sequences of Snail-1/2, Twist-1 and most likely Zeb-2 (although this

has not been documented yet) and are recruited to relieve repressing epigenetic marks and induce

their robust expression. In addition, their restricted expression patterns and functions during

specification clearly link their activity to the acquisition by cells of their neural crest identity

rather than to their delamination. Second, the intracellular localization of Snail-1/2, its stability,

and its activity are dictated by a plethora of kinases and nuclear corepressors which are induced

or activated concurrently just prior to delamination, suggesting that collectively they function as

a molecular switch to induce neural crest cell EMT and that this event requires high doses of

Snail proteins to regulate massively within a short time period a large array of effectors. This

model represents however only a part of the whole picture as it is essentially based on the control

of the repressing activities of Snail proteins on its cadherin targets; and it is now largely

demonstrated that they are also capable of activating numerous genes, in particular those related

to the locomotory machinery.

Spatial control of the activity of the Zeb-2 transcription factor

Contrary to the other core EMT regulatory factors, Zeb-2 is not restricted to premigratory neural

crest cells but is also expressed by the entire neural plate. Zeb-2 expression is induced

precociously during primary neurulation and correlates with the progressive switch from E- to N-

cadherin in neural epithelial cells (Dady et al., 2012). Knock-down of Zeb-2 in the neural tube at

cranial levels in chick or mouse embryos results in persistent expression of E-cadherin in neural

epithelial cells and also in strong inhibition of neural crest cell delamination and migration

(Rogers et al., 2013; Van de Putte et al., 2003). Thus, Zeb-2 is clearly an important player in the

EMT process of neural crest cells by promoting E-cadherin silencing. Yet, unlike neural crest

cells, neural epithelial cells retain their epithelial morphology and fail to undergo EMT despite of

33

persistent Zeb-2 expression over long time periods. Why the same molecule mediates radically-

different behaviors in neighboring cells that share in common almost the same origin and

numerous cellular features? As discussed above, an explanation resides in the fact that neural

tube cells do not express neural crest specifier genes and therefore lack the appropriate signaling

modules that enable them with the ability to undergo full EMT and disperse. However,

additional reasons may be invoked that could account for the exquisite manner by which neural

crest cell delamination is regulated. In particular, it has been found that Zeb-2 is evenly

expressed at moderate levels in the neural tube and neural crest throughout neurulation during

neural crest specification but that its expression is sharply increased in neural crest cells at the

time when they undergo EMT, indicating that execution of the EMT program may require higher

doses of Zeb-2 than the sole control of E- to N-cadherin switch (Dady et al., 2012; Rogers et al.,

2013; Van de Putte et al., 2007). This assumption is supported by a recent study on the genetic

network regulating EMT in cancer cells proposing a model accounting for the three distinct

states in which cells can be alternatively found, i.e. epithelial, mesenchymal and a so-called

hybrid state, in which they show mixed mesenchymal and epithelial characteristics (Lu et al.,

2013). This structural organization of cells was proposed to be regulated by the circuit composed

of the two interconnected modules, the miR-34/Snail-2 and the miR-200/Zeb-2 modules,

mentionned above. Interestingly, this model suggests that the miR-34/Snail-2 module exists only

under two mutually-exclusive forms, (high miR-34/low Snail-2) and (low miR-34/high Snail-2),

and is a central regulatory circuit of EMT, acting as an on/off switch responsible for either the

epithelial or the mesenchymal states. The miR-200/Zeb-2 module in contrast can be found in

three forms, (high miR-200/low Zeb-2), (low miR200/high Zeb-2) and (medium miR-

200/medium Zeb-2), corresponding to the epithelial, mesenchymal and hybrid states,

respectively, and may function as a more-refined modulator of the physiology of epithelial cells.

Although the expression levels of miRNAs in the neural tube remain to be established, the

34

relatively-modest level of Zeb-2 in neural epithelial cells associated with the lack of Snail-2 may

explain why, in spite of E- to N-cadherin switch, these cells undergo only partial transformation

into a pseudostratified epithelium characterized by elongated cell shapes and absence of tight

junctions, but are unable to achieve complete EMT. Thus, the combination of cadherin switches

in the early blastoderm under the tight spatial and temporal control of the core EMT regulatory

factors Snail-2 and Zeb-2 may be responsible for its partition into three distinct portions

exhibiting different epithelial properties and capacities to execute the EMT program.

Future prospects

The few examples described above illustrate how time and space are coordinated in an exquisite

manner to permit a population of progenitor cells to execute sequentially each of the individual

steps that eventually lead them to populate a great variety of territories in the embryo and

provide an extremely large array of cell types. This is achieved through an intricate combination

of genome-wide epigenetic control of gene expression, recruitment of specific transcription

factors that act in concert and activation of chemical modifications that regulate locally the

availability and activity of these factors. How these different actors are timely coordinated and

whether there is an hierarchy among them will certainly constitute an important subject of

investigation in the near future. Clearly the sole activations of signaling pathways such as BMPs,

Wnt and FGF are far from sufficient to account for the precision by which these events occur, as

illustrated in the case of induction of the neural plate border. Physical processes such as

constraints applied by forces exerted by moving cells or the viscoelastic properties of tissues are

likely to play instructive roles at the scale of the whole embryo. We are only starting to address

the question of how these physical processes are translated into biological cascades and vice

versa how biological mechanisms can modulate and refine ordered and coherent physical events.

35

Acknolwledgments

The authors wish to thank Paul Trainor for his kind invitation to write this review and for his

patience and to express their friendship for all their colleagues working in the field for their

endless imagination and stimulating contributions.

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