Developmental Biology 389 (2014) 98–119

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Developmental Biology
journal homepage: www.elsevier.com/locate/developmentalbiology

The evolutionary history of vertebrate cranial placodes II. Evolution

of ectodermal patterning
Gerhard Schlosser a,n, Cedric Patthey b,1, Sebastian M. Shimeld b
a
Department of Zoology, School of Natural Sciences & Regenerative Medicine Institute (REMEDI), National University of Ireland,
University Road, Galway, Ireland
b
Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK

art ic l e i nf o a b s t r a c t

Available online 1 February 2014 Cranial placodes are evolutionary innovations of vertebrates. However, they most likely evolved by
Keywords: redeployment, rewiring and diversification of preexisting cell types and patterning mechanisms. In the
Placodes second part of this review we compare vertebrates with other animal groups to elucidate the
Six1 evolutionary history of ectodermal patterning. We show that several transcription factors have ancient
Eya bilaterian roles in dorsoventral and anteroposterior regionalisation of the ectoderm. Evidence from
BMP amphioxus suggests that ancestral chordates then concentrated neurosecretory cells in the anteriormost
Wnt non-neural ectoderm. This anterior proto-placodal domain subsequently gave rise to the oral siphon
Preplacodal ectoderm primordia in tunicates (with neurosecretory cells being lost) and anterior (adenohypophyseal, olfactory,
Neural crest
and lens) placodes of vertebrates. Likewise, tunicate atrial siphon primordia and posterior (otic, lateral
Amphioxus
line, and epibranchial) placodes of vertebrates probably evolved from a posterior proto-placodal region
Ciona
Neural induction in the tunicate–vertebrate ancestor. Since both siphon primordia in tunicates give rise to sparse
populations of sensory cells, both proto-placodal domains probably also gave rise to some sensory
receptors in the tunicate–vertebrate ancestor. However, proper cranial placodes, which give rise to high
density arrays of specialised sensory receptors and neurons, evolved from these domains only in the
vertebrate lineage. We propose that this may have involved rewiring of the regulatory network upstream
and downstream of Six1/2 and Six4/5 transcription factors and their Eya family cofactors. These proteins,
which play ancient roles in neuronal differentiation were first recruited to the dorsal non-neural
ectoderm in the tunicate–vertebrate ancestor but subsequently probably acquired new target genes in
the vertebrate lineage, allowing them to adopt new functions in regulating proliferation and patterning
of neuronal progenitors.
& 2014 Elsevier Inc. All rights reserved.

Introduction supporting cells of the olfactory and vomeronasal epithelia and

During their adoption of a more active and predatory life style,
stem vertebrates evolved a “new head” with highly specialised
musculoskeletal and neurosensory structures (Northcutt and Gans,
1983). Most of these structures are derived from only two novel
embryonic tissues, the neural crest and the cranial placodes. As
summarised in the first part of this review (Patthey et al., in this
issue; see also Baker and Bronner-Fraser, 2001; Grocott et al.,
2012; Schlosser, 2010) the latter make important contributions to
many sense organs and cranial ganglia of the vertebrate head.
Olfactory placodes develop into chemosensory receptor and
n
Corresponding author.
E-mail address: gerhard.schlosser@nuigalway.ie (G. Schlosser).
1
Present address: Umeå Center for Molecular Medicine, Building 6M, 4th floor,
Umeå University, 901 87 Umeå, Sweden.
also form neurosecretory (e.g. GnRH) cells migrating into the
forebrain. The lens placode develops into the lens of the eye. Otic
and lateral line placodes give rise to the mechanosensory hair cells
and their supporting cells in the inner ear and the lateral line
system, respectively as well as to the sensory neurons innervating
them. The epibranchial placodes produce viscerosensory neurons
in the ganglia of the facial, glossopharyngeal, and vagal nerve,
while the profundal and trigeminal placodes (ophthalmic and
maxillomandibular placodes in amniotes) give rise to somatosen-
sory neurons of the trigeminal nerve. Finally, the adenohypophy-
seal placode develops into the anterior pituitary, the major
hormonal control organ of vertebrates with six classes of
endocrine cells.
Insights into the evolutionary history of placodes come mostly
from comparisons with other living taxa, since the information
that can be obtained from fossils is limited. Their phylogenetic

http://dx.doi.org/10.1016/j.ydbio.2014.01.019
0012-1606 & 2014 Elsevier Inc. All rights reserved.
 G. Schlosser et al. / Developmental Biology 389 (2014) 98–119
relationships were described in the first part of our review
((Patthey et al., in this issue); see also Fig. 6 below). To summarise
this briefly, tunicates (including ascidians, appendicularians and
thaliaceans) are now known to be the chordates most closely
related to vertebrates, with cephalochordates (amphioxus) being
more distantly related (Delsuc et al., 2006). Together with the
ambulacrarians (hemichordates and echinoderms) the chordates
form the deuterostomes. Deuterostomes and protostomes (ecdy-
sozoans and lophotrochozoans) comprise the bilaterians; and the
latter together with the cnidarians and the more distantly related
sponges form the metazoans. Although tunicates have evolved a
highly specialised mode of development (Lemaire, 2009; Lemaire
et al., 2008), they share some derived aspects of embryonic
development with vertebrates. These allow us to some extent to
infer how the tunicate–vertebrate ancestor developed, whereas
characters shared with amphioxus give insights into the ancestral
chordate condition and characters shared with other animal groups
can be traced to even more distant ancestors.
The evolution of placodes has attracted a lot of debate and
in the first part of our review (Patthey et al., in this issue) we have
provided a brief historical overview of various scenarios proposed.
We also emphasised there that even though cranial placodes and
the placode-derived sense organs are evolutionary innovations of
vertebrates, they most likely evolved by redeployment and rewir-
ing the gene regulatory networks of preexisting cell types and
patterning mechanisms. It may, therefore, be possible to recognise
homologues of placodal cell types and of placodal patterning
mechanisms in invertebrates, despite the fact that proper cranial
placodes evolved as novel structures in the vertebrate lineage (and
as such have no invertebrate homologue). We have discussed the
evolutionary history of placodal cell types in the first part of this
review (Patthey et al., in this issue). In the second part we will now
focus on the evolution of ectodermal patterning mechanisms. We
will first give a brief overview of placode development in verte-
brates considering them within the broader context of ectodermal
patterning along both the dorsoventral and anteroposterior axis.
We will then discuss how ectodermal patterning mechanisms
along the dorsoventral and anteroposterior axis were modified
during placode evolution. We conclude by suggesting future
research challenges and opportunities.
Placodes and ectodermal patterning in vertebrates
All placodes originate from a common domain, the preplacodal
ectoderm (or panplacodal primordium) and hence from an ecto-
dermal territory that has a well-defined position and molecular
address along the dorsoventral and anteroposterior axes (Fig. 1).
To understand the evolutionary origin of placodes, it is therefore
important to consider how the distinct identity of this territory is
specified in vertebrates and how the underlying dorsoventral and
anteroposterior patterning mechanisms evolved. Because mechan-
isms of early ectodermal patterning in vertebrates have been
reviewed extensively elsewhere (De Robertis, 2009; Grocott
et al., 2012; Groves and LaBonne, in this issue; Milet and
Monsoro-Burq, 2012; Niehrs, 2010; Patthey and Gunhaga, 2011;
Saint-Jeannet and Moody, in this issue; Sauka-Spengler and
Bronner-Fraser, 2008; Schier and Talbot, 2005; Schlosser, 2010;
Stern, 2006; Vonica and Brivanlou, 2006), we will here provide
only a brief overview.
The dorsoventral axis
The preplacodal ectoderm develops as a crescent-shaped
region surrounding the anterior neural plate and sandwiched in
between the neural plate and the epidermis. Rostrally, the
99
preplacodal ectoderm directly abuts the neural plate, but caudally
the neural crest is intercalated between preplacodal ectoderm and
neural plate (Fig. 1). This arrangement of ectodermal territories is
set up during gastrula and early neurula stages, when signals from
the ectoderm itself and adjacent tissues induce the dorsoventrally
restricted expression of various transcription factors, which in turn
specify different ectodermal fates in a combinatorial fashion. We
have in recent years obtained many new insights into how this
process is regulated although some controversies remain and
details are still being worked out.
Unquestionably, the subdivision of the ectoderm is closely tied
to the process of neural induction. This involves a number of
different signalling molecules including Wnts, FGFs and inhibitors
of BMPs that sequester BMPs dorsally thereby creating a ventral to
dorsal gradient of free BMPs. BMPs are then thought to act as
morphogens, which activate different target genes in a concentra-
tion dependent manner (Neave et al., 1997; Nguyen et al., 1998;
Schumacher et al., 2011; Tozer et al., 2013; Wilson et al., 1997).
It has been proposed that the four main ectodermal territories –
epidermis, preplacodal ectoderm, neural crest and neural plate –
are set up in direct response to different BMP concentrations (e.g.
Brugmann et al., 2004; Glavic et al., 2004; Tribulo et al., 2003).
However, recent evidence suggests a more complex picture, in
which neural crest and preplacodal ectoderm each require a
cocktail of inducing signals and have different requirements for
BMP signalling at different times (Ahrens and Schlosser, 2005;
Kwon et al., 2010; Litsiou et al., 2005; Patthey et al., 2009; Reichert
et al., 2013; Steventon et al., 2009).
An important unresolved question is, whether neural crest and
preplacodal ectoderm arise from a common ectodermal precursor
region or whether they have independent developmental origins.
According to the first model (neural plate border state model),
ectoderm in between epidermis and neural plate is initially
specified as “neural plate border” and will subsequently be
induced to form neural crest or preplacodal ectoderm depending
on signals present (Brugmann et al., 2004; Glavic et al., 2004;
Litsiou et al., 2005; Meulemans and Bronner-Fraser, 2004). Accord-
ing to the second model (binary competence model), two ecto-
dermal competence territories – neural and non-neural – are
initially specified during gastrulation, with preplacodal ectoderm
being subsequently induced from the non-neural territory and
neural crest from the neural territory (Ahrens and Schlosser, 2005;
Pieper et al., 2012). Since dorsal restriction of neural and ventral
restriction of non-neural competence is only established during
gastrulation, even the latter model is compatible with the tran-
sient formation of an unspecified neural plate border region as has
been pointed out recently (Grocott et al., 2012). However, both
models imply different hierarchies of lineage decisions. The neural
plate state border model predicts lineage restrictions in the
sequence [neural plate–neural plate border (neural crest–prepla-
codal)–epidermis], whereas the binary competence model predicts
a sequence [neural (neural plate–neural crest)–non-neural (epi-
dermis–preplacodal)]. Additional experiments involving single cell
lineage analysis are required to resolve this issue.
Spatiotemporal analysis of expression patterns in combination
with functional studies suggest that the combinatorial activity of a
large number of transcription factors and signalling molecules is
involved in the specification of the neural crest and the preplaco-
dal ectoderm. Neural crest specifiers such as Sox9/10, FoxD3,
Snail1/2, Twist, c-Myc, and AP2 define the neural crest domain
and control neural crest migration and cytodifferentiation
(reviewed in Betancur et al., 2010; Meulemans and Bronner-
Fraser, 2004; Milet and Monsoro-Burq, 2012; Sauka-Spengler and
Bronner-Fraser, 2008). Conversely, transcription factors Six1 and
Six4 appear to play a central role in defining the preplacodal
ectoderm (reviewed in Grocott et al., 2012; Schlosser, 2010). They
100 G. Schlosser et al. / Developmental Biology 389 (2014) 98–119

Fig. 1. Dorsoventral ectodermal patterning and induction of panplacodal primordium. (A) Two models have been proposed. According to the “neural plate border state
model”, neural crest and panplacodal primordium develop from a common neural plate border region, while according to the “binary competence model”, neural crest is
induced from a neural competence territory (green) and panplacodal primordium from a non-neural competence territory (yellow). (B) Time course of induction according to
the binary competence model. During gastrulation dorsally enriched neural competence factors (green) and ventrally enriched non-neural competence factors (yellow) are
overlapping (left panel) but distinct competence territories are established at the end of gastrulation (middle panel). Neural crest inducing signals (FGF, Wnt, and BMP; blue)
then induce neural crest at the border of the neural territory, while other signals (FGF, BMP- and Wnt-inhibitors; red) induce the panplacodal primordium at the border of
the non-neural ectoderm (right panel) (modified from Schlosser, 2006).

act in a complex with coactivators of the Eya family. However, the
latter also have phosphatase activity and probably play additional
important roles in the cytoplasm, which may be independent of
Six (reviewed in Jemc and Rebay, 2007; Tadjuidje and Hegde,
2013). Ectodermal expression of Six1, Six4 and Eya family mem-
bers is restricted to the preplacodal ectoderm from neural plate
stages onwards but coexpression of Six and Eya family members is
also observed in a number of other structures including the
developing pharyngeal pouches, somites and kidneys. In the
cranial ectoderm, Six1 and Eya are essential not only for the
expression of other preplacodal marker genes early on (Brugmann
et al., 2004; Christophorou et al., 2009), but also for subsequent
development of various placodes. Six1 and Eya promote both the
proliferation of neuronal progenitors and sensory and neuronal
differentiation, and are also required for proper morphogenesis of
placodes (e.g. Ahmed et al., 2012a, 2012b; Kozlowski et al., 2005;
Laclef et al., 2003; Schlosser et al., 2008; Xu et al., 1999; Zheng
et al., 2003; Zou et al., 2004). Indeed some cell cycle control genes,
the hair cell determination gene Atoh1 and cytoskeletal modula-
tors such as Ezrin have been identified as direct Six target genes,
which may mediate some of these effects, but much remains to be
learned about the precise mechanisms and other targets involved
(e.g. Ahmed et al., 2012b; Li et al., 2003; Yu et al., 2006).
In vivo experiments have shown that the preplacodal domain
of Six and Eya expression is induced at neural fold stages by
signals from the underlying mesoderm and adjacent neural plate,
which include FGFs and inhibitors of the Wnt pathway (Ahrens
and Schlosser, 2005; Brugmann et al., 2004; Litsiou et al., 2005).
BMP inhibits expression of Six1, Six4 and Eya2 in posterior
preplacodal ectoderm at these stages (Ahrens and Schlosser,
2005; Litsiou et al., 2005), suggesting that BMP-inhibition at
neural fold stages is also required for induction and/or mainte-
nance of the preplacodal ectoderm, at least in its posterior part.
In contrast, an in vitro study showed that BMP did not block
anterior Six1 expression at neural fold stages and even suggested
that the induction of the preplacodal ectoderm at anterior levels is
mediated by BMP signals at the late gastrula stage (Sjödal et al.,
2007). Therefore, further studies are needed to clarify the role of
BMP-inhibition for induction of anterior preplacodal ectoderm
in vivo. Neural crest induction, which is already drawing to a close
at neural fold stages instead requires Wnt, FGF and at least low
level BMP signalling (Basch et al., 2000; Mancilla and Mayor, 1996;
Monsoro-Burq et al., 2003; Patthey et al., 2009; Reichert et al.,
2013; Steventon et al., 2009).
It is still controversial whether ectoderm at the neural plate
border is competent to give rise to both neural crest and placodes
as the “neural plate border state model” proposes or whether both
derive from different ectodermal territories as the “binary compe-
tence model” predicts (Fig. 1A). Several ectodermal transcription
factors establish dorsally or ventrally restricted expression
domains during gastrulation and are, therefore, candidate regula-
tors of neural vs. non-neural competence, respectively (Fig. 1B).
Dorsally (neurally) enriched transcription factors include SoxB1
(Sox2 and Sox3), Geminin and Zic family members, while ventrally
restricted transcription factors include Dlx3/Dlx5, Msx1, GATA2/3,
AP2, FoxI and Vent family members (reviewed in Betancur et al.,
2010; Grocott et al., 2012; Groves and LaBonne, in this issue; Saint-
Jeannet and Moody, in this issue; Schlosser, 2010). However, the
precise dorsoventral extent of the expression domains of these
various transcription factors differs and dorsally and ventrally
restricted transcription factors initially overlap extensively at the
neural plate border region. During gastrulation, expression
domains gradually become more restricted and form sharper
boundaries possibly by cross-repressive interactions between
transcription factors. At the end of gastrulation, at least one of
the Dlx proteins (Dlx5 or Dlx3, depending on the species), GATA2/
3 and FoxI are excluded from the prospective neural and neural
crest territories, while SoxB1 factors become restricted to the
 G. Schlosser et al. / Developmental Biology 389 (2014) 98–119
neural plate and Zic family members to the lateral neural plate and
neural crest. Msx1, Vent and AP2 maintain relatively broad
expression from the epidermis to neural crest or even lateral
neural plate (Msx1). Several other transcription factors (c-Myc, Id,
Hairy2, Irx1, and Pax3) are established in more narrow domains
that cover neural crest and lateral neural plate territories but may
extend into preplacodal ectoderm as well (c-Myc, Id, Hairy2, and
Irx1) (see reviews by Betancur et al., 2010; Grocott et al., 2012;
Schlosser, 2010).
Recent studies in zebrafish and Xenopus suggest that Dlx3,
GATA2/3, FoxI and AP2 in the non-neural ectoderm cross-activate
each other, cooperate in the promotion of non-neural competence
and are cell-autonomously required in conjunction with the
proper signals for induction of preplacodal markers even though
there may be some differences between species in their relative
importance (Bhat et al., 2012; Kwon et al., 2010; Pieper et al.,
2012). Whether dorsally restricted transcription factors like Sox3
and Geminin form a similar network that promotes neural
competence remains to be shown. In the lateral part of the neural
ectoderm, AP2 plays a different role than in non-neural ectoderm
and cooperates with Msx1, Zic1 and Pax3 to promote the forma-
tion of neural crest at the expense of neural plate cells (Hong and
Saint-Jeannet, 2007; Li and Cornell, 2007; Luo et al., 2002; Milet
et al., 2013; Monsoro-Burq et al., 2005).
But how are the dorsally and ventrally restricted expression
domains of neural and non-neural competence factors set up in
the first place? Recent findings show that the preplacodal
ectoderm and epidermis can only be induced in ectoderm
exposed to BMP during early gastrulation (Kwon et al., 2010),
whereas induction of neural plate and neural crest requires
absence of BMP activity during that time (Patthey et al., 2009;
Steventon et al., 2009). This suggests that the BMP gradient
established during gastrulation probably plays a crucial role in
setting up dorsally and ventrally restricted expression domains of
neural and non-neural competence factors, respectively. Indeed
most genes with ventrally restricted expression are activated by
BMP either directly (e.g. Msx1, GATA2/3, and Vent) or indirectly,
before their expression becomes BMP-independent at the end of
gastrulation, whereas genes with dorsally restricted expression
are repressed by BMP (Feledy et al., 1999; Friedle and Knöchel,
2002; Kwon et al., 2010; Mizuseki et al., 1998; Pera et al., 1999;
Suzuki et al., 1997).
The anteroposterior axis
The preplacodal ectoderm develops only in the head of verte-
brates and different placodes develop from different subregions of
the preplacodal ectoderm depending on their position along the
anteroposterior axis. There is now increasing evidence that ante-
roposterior patterning of placodes is linked to general anteropos-
terior patterning mechanisms of the ectoderm (Grocott et al.,
2012; Schlosser, 2006, 2010). Wnt, retinoic acid (RA) and FGF
signals in particular have been shown to act as posteriorising
factors in the early (gastrula to neurula) vertebrate embryo
(Altmann and Brivanlou, 2001; Hikasa and Sokol, 2013; Niehrs,
2010; Schier and Talbot, 2005). Wnt antagonists are released from
cranial tissues, sequester Wnts in the head and help to establish a
Wnt gradient that patterns the ectoderm as well as other germ
layers along the anteroposterior axis in a concentration dependent
fashion via the canonical Wnt signalling pathway (Kiecker and
Niehrs, 2001; Niehrs, 2010). Both Wnts and retinoic acid have been
implicated in repressing preplacodal ectoderm formation in the
trunk thereby setting its posterior boundary (Brugmann et al.,
2004; Janesick et al., 2012; Litsiou et al., 2005).
In response to these signals, anteroposteriorly restricted
expression domains of several transcription factors, which direct
101
Fig. 2. Anteroposterior ectodermal patterning and origin of individual placodes
from the panplacodal primordium. The panplacodal primordium is patterned along
the anteroposterior axis by posteriorly restricted Wnt signals and by various
signalling centres in the neural plate and mesoderm. These induce transcription
factors within the panplacodal primordium (red), which specify precursor regions
for multiplacodal areas (coloured outlines) and individual placodes (coloured
ovals). A few examples of transcription factors are given below. Ad: adenohypo-
physeal placode; ANR: anterior neural ridge; EB: epibranchial placodes; EF: eye
field; L: lens placode; LL: lateral line placodes; MHB: midbrain–hindbrain bound-
ary; Not: notochord; Ol: olfactory placode; Ot: otic placode; PP: pharyngeal
pouches, Pr/V: profundal/trigeminal placode; R4: rhombomere 4 (modified from
Schlosser, 2010).
ectodermal fates in a region-specific manner are set up in the
ectoderm during gastrulation (Fig. 2). For example, the direct Wnt
targets Cdx, Meis, Gbx and Irx are activated posteriorly but with
different anterior expression limits and these in turn activate
further posterior transcription factors such as Hox proteins
(Elkouby et al., 2010; Gomez-Skarmeta et al., 2001; Kiecker and
Niehrs, 2001; Li et al., 2009; Nordström et al., 2006; Pilon et al.,
2006). Subsequently, mutual repression between posteriorly
restricted and anteriorly restricted factors such as Gbx and Otx,
Pax2 and Pax6, Irx and Six3 or Irx and Fezf, respectively establishes
sharp boundaries between transcription factor domains in the
neural plate (Broccoli et al., 1999; Kobayashi et al., 2002; Martinez-
Barbera et al., 2001; Millet et al., 1999; Rodriguez-Seguel et al.,
2009). However, most of these anteroposteriorly restricted tran-
scription factors are not confined to the neural ectoderm suggest-
ing that similar interactions may regionalise the non-neural
ectoderm as well. This has recently been confirmed for Gbx2 and
Otx2, which mutually repress each other in non-neural ectoderm
and are required for development of posterior (otic and epibran-
chial) and anterior (olfactory, lens, and trigeminal) placodes,
respectively (Steventon et al., 2012).
Various signalling centres are then established at expression
boundaries between different transcription factors in the neural
plate or in the underlying mesoderm such as the midbrain–
hindbrain boundary, which develops where Gbx2 and Otx2 are
juxtaposed (Broccoli et al., 1999; Millet et al., 1999). Signals from
these centres locally induce new transcription factors in both the
neural and non-neural ectoderm resulting in their more fine-
grained subdivisions (Fig. 2). Due to these processes, a complex
pattern of transcription factor domains is established in the
preplacodal ectoderm during gastrulation and neurulation, which
promote the formation of different placodes in a multistep and
combinatorial fashion. It has become increasingly clear that during
102 G. Schlosser et al. / Developmental Biology 389 (2014) 98–119
this process relatively broad multiplacodal areas are established
first, in which individual placodes are specified only later
(reviewed in Grocott et al., 2012; Schlosser, 2006, 2010).
The extended anterior placodal area, which expresses Pitx,
Six3, Pax6, FoxE, and Dmrt (as well as Anf and FoxG1 in its more
anterior part) in response to neuropeptides (somatostatin and
nociceptin) from underlying mesoderm and the rostral preplaco-
dal ectoderm itself (Lleras-Forero et al., 2013) will form adenohy-
pophyseal, olfactory and lens placodes in a signalling dependent
manner (reviewed in Grocott et al., 2012; Schlosser, 2010; Toro and
Varga, 2007). For example, hedgehog signalling from midline
tissues favors adenohypophyseal over lens fates, while extended
time of exposure to BMP signals promotes lens over olfactory fates
and FGF signalling from the anterior neural plate promotes
olfactory over lens fates (Bailey et al., 2006; Dutta et al., 2005;
Karlstrom et al., 1999; Sjödal et al., 2007; Treier et al., 2001). The
posterior placodal area, which expresses Pax2, Pax8, Sox3, and
Sox2 among other transcription factors and is induced by FGF
signals from adjacent neural plate and mesoderm will give rise to
otic, epibranchial and lateral line placodes (reviewed in Grocott
et al., 2012; Ladher et al., 2010; Schlosser, 2010). Subsequently,
persistent FGF signals promote epibranchial over otic fates in this
domain, whereas Wnt signals from neural plate and/or mesoderm
favour otic over epibranchial fates (Freter et al., 2008; Ladher et al.,
2000; Nechiporuk et al., 2007; Ohyama et al., 2006). Finally, there
are the profundal and trigeminal placodes (ophthalmic and max-
illomandibular trigeminal placodes of amniotes, respectively), the
former characterised by Pax3 expression, whereas no unique
marker has yet been identified for the latter (reviewed in
Grocott et al., 2012; Schlosser, 2010). These placodes, which are
sandwiched between the anterior and posterior group of placodes,
are induced by a combination of signals (Wnt, FGF, and PDGF)
from the neural tube (Canning et al., 2008; Lassiter et al., 2007;
McCabe and Bronner-Fraser, 2008).
The evolution of dorsoventral ectodermal patterning
We will now consider, when distinct ectodermal territories,
which subsequently became precursors for neural crest and
placodes first evolved, and how the mechanisms positioning these
territories along the dorsoventral axis changed in the course of
evolution. The answer to this question is probably complex since
the emergence of dorsoventral patterning mechanisms may be
more ancient than the establishment of neural and non-neural
ectodermal territories, and the latter in turn may precede the
origin of specialised border territories in the neural and non-
neural ectoderm and formation of proper neural crest and pla-
codes within these respective domains. We, thus, will consider
these different steps in turn here.
Origin of dorsoventral patterning mechanisms
Comparative data from many taxa including representatives of
lophotrochozoans (e.g. Platynereis), ecdysozoans (e.g. Drosophila)
and deuterostomes (hemichordates, cephalochordates, and verte-
brates) indicate that a gradient of BMP signalling established by
activating BMPs and their antagonists on opposite sides of the body
plays an ancient role in patterning the dorsoventral axis in
bilaterians (Akiyama-Oda and Oda, 2006; Denes et al., 2007;
Ferguson and Anderson, 1992; Holley et al., 1995; Lowe et al.,
2006; Mizutani et al., 2005; Molina et al., 2007; Sasai et al., 1995;
van der Zee et al., 2006; Yu et al., 2007). This patterning mechanism
was secondarily lost in a few groups including tunicates (Darras and
Nishida, 2001a, 2001b; Lemaire et al., 2008) and was modified in
echinoderms, where a BMP gradient forms despite colocalisation of
BMP with chordin on the ventral side (Lapraz et al., 2009). However,
it should be noted that coexpression of BMPs with BMP antagonists
is also observed on both ventral and dorsal sides in vertebrates but
these are under opposite transcriptional regulation (Reversade and
De Robertis, 2005). Moreover, ventral enrichment of proteases (e.g.
Tolloid) which cleave chordin, promotes shuttling of BMP by
chordin to the ventral side of the embryo. These complex regulatory
interactions, many of which also appear to be evolutionarily
conserved, endow the BMP gradient with self-regulatory properties
(De Robertis, 2009; Eldar et al., 2002; Reversade and De Robertis,
2005).
Asymmetric expression of BMPs and BMP antagonists along
one body axis (the so-called directive axis orthogonal to the oral–
aboral axis) is already observed in some cnidarians and has been
shown to be involved in BMP gradient formation even though BMP
and its antagonists are colocalised (Matus et al., 2006; Rentzsch
et al., 2006; Saina et al., 2009), suggesting that the use of BMP
gradients in patterning one of the body axes may predate
bilaterians. Since BMP antagonists are expressed on the ventral
(i.e. mouth-forming) side in all protostomes as well as in hemi-
chordates, but are confined to the dorsal side in chordates, the
dorsoventral axis was probably inverted in the chordate ancestor
accompanied by the formation of a new mouth (Arendt and
Nübler-Jung, 1994). It is, however, still controversial how this
was achieved and to what extent target genes and structures
downstream of the BMP morphogen gradient were preserved in
evolution.
We will now discuss this latter aspect more closely focussing
on the ectoderm. Both in arthropods and in chordates BMP
morphogen gradients were shown to be important during two
phases of partitioning the ectoderm (Dorfman and Shilo, 2001;
Kwon et al., 2010; Lu et al., 2012; Rusten et al., 2002). In the early
phase, complementary neural and non-neural ectodermal terri-
tories are established. The neural ectoderm, which will form the
central nervous system (CNS) and parts of the peripheral nervous
system (PNS; e.g. ventral sense organs in arthropods and neural
crest in vertebrates), requires low levels of BMP signalling, while
the non-neural ectoderm, which will give rise to epidermis and
other parts of the PNS (dorsolateral sense organs in arthropods
and placodes in vertebrates), requires high levels (Akiyama-Oda
and Oda, 2006; Arora et al., 1994; Biehs et al., 1996; Holley et al.,
1995; Lu et al., 2012; Sasai et al., 1995; van der Zee et al., 2006).
Additional signals may contribute to neural induction in some taxa
(e.g. FGF in tunicates and vertebrates) but these are not conserved
throughout bilaterians (Bertrand et al., 2011; Huang and Stern,
2005). In the later phase, the position of the neural/non-neural
boundary has been set but a BMP gradient helps to further
subdivide the neural territory and establish several molecularly
distinct domains of transcription factor expression, which subse-
quently give rise to distinct neuron types (Dickinson et al., 1995;
Liem et al., 1997; Liem et al., 1995; Mizutani et al., 2006; von Ohlen
and Doe, 2000). In Drosophila, a BMP gradient has also been
implicated in further subdivision of the non-neural ectoderm
(Rusten et al., 2002; Sutherland et al., 2003).
Because in arthropods, annelids and chordates, the CNS forms
from ectoderm exposed to lowest levels of BMP signalling and its
formation is suppressed by BMP, it has initially been suggested
that BMP has an anti-neurogenic effect (Arendt and Nübler-Jung,
1997; De Robertis and Sasai, 1996). However, pan-neuronal mar-
kers such as Elav are not affected by elevated levels of BMP
signalling in either annelids or hemichordates (Denes et al.,
2007; Lowe et al., 2006), whereas in amphioxus elevated BMP
early on represses Elav expression in the neural plate but at later
stages causes its expansion in the non-neural ectoderm, (Lu et al.,
2012). This has led to the revised proposal that BMP only represses
the motor- and interneurons of the CNS but promotes formation of
 G. Schlosser et al. / Developmental Biology 389 (2014) 98–119 103

sensory neurons in the PNS (Arendt et al., 2008). However, there is
presently little evidence that the BMP gradient acts by promoting
or repressing different cell types directly. More likely, it acts by
specifying a series of transcription factor domains, which then
serve as molecular addresses along the dorsoventral axis (e.g.
Niehrs, 2010). Since most of these expression domains give rise to
multiple cell types, at least some of these transcription factors may
function as competence factors, which are required for the
formation of a set of cell types but require additional input (e.g.
from signalling pathways and other transcription factors with
anteroposteriorly restricted domains of expression) to specify a
particular cell type. In the following, we will first consider how the
domains of transcription factors providing neural and non-neural
ectodermal competence in vertebrates in response to an early BMP
gradient evolved. We will then review how transcription factor
domains that specify distinct border identities within the neural
and non-neural territories originated in evolution.
Origin of neural vs. non-neural ectoderm
Chordates
A dorsal neural plate is one of the defining features of the
chordates and many of the transcription factors implicated in the
formation of neural or non-neural ectoderm in vertebrates show
similar expression patterns in amphioxus and tunicates (Fig. 3).
In amphioxus, the expression domains of these transcription
factors are set up during gastrulation in response to a BMP
gradient similar to vertebrates (Kozmikova et al., 2011, 2013; Yu
et al., 2007, 2008), but in tunicates upstream mechanisms have
greatly diverged (Lemaire, 2009).
Similar to vertebrates, SoxB1 and Zic genes in amphioxus
become restricted to the dorsal (neural) ectoderm during gastrula-
tion, while AP2, Dlx, Msx, and Vent genes become restricted to the
ventral ectoderm (Gostling and Shimeld, 2003; Holland et al.,
1996, 2000; Kozmik et al., 2001; Meulemans and Bronner-Fraser,
2002, 2007; Sharman et al., 1999; Yu et al., 2007, 2008). In contrast
to vertebrates, amphioxus GATA1/2/3 is not expressed in the early
ectoderm (Zhang and Mao, 2009), while the expression of the
amphioxus FoxI or Geminin genes has not been determined. The
precise expression boundaries of ventral ectodermal genes in
amphioxus differ and only AP2 appears to be restricted to non-
neural ectoderm, whereas Dlx is also expressed at the very edge of
the anterior neural plate and Msx and Vent extend further into the
neural plate. However, functional data are missing, and it remains
to be determined which cross-regulatory interactions among
transcription factors are essential for establishing the boundary
between neural and non-neural ectoderm in amphioxus. Never-
theless, these expression patterns suggest that neural and non-
neural competence territories in amphioxus may be overall
specified in a similar fashion to vertebrates, indicating that this
mechanism of specification was already present in the chordate
ancestor.
In ascidians, early pattern formation mechanisms have become
modified drastically and rely heavily on local cell interactions or on

Fig. 3. Schematic overview of transcription factor expression domains and origin of differentiated cell types around the neural plate in chordate embryos. The neural plate is
indicated in green and epidermis in yellow. Domains of Six1/2 and Eya expression are shown in red. Pale red domains in amphioxus (Hatschek's pit and sensory cells) and
tunicates (atrial siphon primordia) indicate expression domains only established at later developmental stages. Coloured outlines enclose transcription factor expression
domains except for Msx1 in all taxa and FoxI in vertebrates, which are expressed peripheral to the lines shown. Hatched lines indicate transcription factor expression
domains that are only established at later developmental stages. The Snail1/2 expression domains in amphioxus and tunicates are shown by a blue outline, whereas the
neural crest domain in vertebrates, which coexpresses Snail1/2 with an array of other transcription factors is shown in solid blue. Expression domains of Irx and Gbx genes
are not shown, but are posteriorly abutting the domains of Six3/6 and Otx expression, respectively. Some expression domains are found only in a subset of taxa as indicated
(A: amphioxus; T: tunicates; V: vertebrates). See text for details.
104 G. Schlosser et al. / Developmental Biology 389 (2014) 98–119
lineage dependent segregation of cell fate determinants rather
than on morphogens (Lemaire, 2009; Lemaire et al., 2008). This is
also true for the segregation of neural and non-neural fates in the
ectoderm, which rely on different mechanisms in the anterior
neural plate (derived from the a-blastomere at 8-cell stage
(Nishida, 1987)) and the posterior neural plate (derived from the
A- and b-blastomeres at 8-cell stage (Nishida, 1987)). The A-line
precursors of the posterior neural plate appear to be cell-
autonomously specified as neural, but do retain competence to
form dorsal mesoderm when exposed to FGF until about the 64-
cell stage (Hudson et al., 2003; Minokawa et al., 2001). In contrast,
the a-line precursors of the anterior neural plate give rise only to
ectodermal fates and become induced as neural in a number of
steps (Lemaire, 2009; Lemaire et al., 2002).
The maternal transcription factor GATA4/5/6 is essential for
ectoderm formation in a-line cells and together with the maternal
factor ETS promotes neural fates in the presence of FGF, and non-
neural fates in its absence. As a consequence, the early neural
marker Otx (Wada et al., 1996b) is activated only in those a-line
cells (a6.5) bordering the FGF9/16/20 expressing A-line cells at the
32-cell stage (Bertrand et al., 2003; Hudson et al., 2003; Hudson
and Lemaire, 2001; Rothbächer et al., 2007). Descendants of this
cell give rise to two rows of cells at the 110-cell stage (rows V/VI
and III/IV), which will divide once more to form the anteriormost
4 rows of cells in the so-called “neural plate” of ascidians (Fig. 4).
However, only the posterior of these two rows (row III/IV) will
ultimately contribute to the CNS, while the anterior row (row
V/VI) will form epidermis and palps. The maintenance of neural
fate in the posterior row and their suppression in the anterior row
depends on the action of FGF signals from the adjacent A-line cells
which inhibit expression of FoxC and promote expression of the
Ciona Zic homologue ZicL in the posterior row (Wagner and
Levine, 2012). At earlier developmental stages, precocious ZicL
expression and neural fate specification in the bipotential palp-
neural precursor cells is prevented by active transcriptional
repression of ZicL in these precursors (Ikeda et al., 2013).
Similar to vertebrates, Zic expression in Ciona defines the
neural ectoderm at the end of gastrulation together with SoxB1,
but both genes are then quickly downregulated suggesting that
other transcription factors are required for maintaining neural
fates (Imai et al., 2002, 2004, 2006; Miya and Nishida, 2003; Wada
and Saiga, 2002; Wagner and Levine, 2012). Also similar to
vertebrates, one of the three Ciona Dlx genes, the Dlx2/3/5 homo-
logue DllB, is confined to the non-neural ectoderm including rows
V and VI at this stage, and AP2 and GATA1/2/3 are widely expressed
in non-neural ectoderm although AP2 expression does not extend
into rows V and VI and the precise boundary of GATA1/2/3 at the
neural plate border is not known (Imai et al., 2004, 2006; Irvine
et al., 2007; Wada et al., 1999). Moreover, there is some evidence
that DllB and GATA1/2/3 may cross-activate each other and that
DllB is required for activating markers of epidermis and palps (Imai
et al., 2006; Irvine et al., 2011). Also Msx in Ciona is expressed in
epidermis and lateral neural plate (Imai et al., 2004) although
expression of another Msx gene in Molgula extends throughout the
neural plate (Ma et al., 1996). Unlike in vertebrates, FoxI is not
expressed in the general non-neural ectoderm (Mazet et al., 2005)
and Vent is lost from the Ciona genome (Wada et al., 2003).
However, different from vertebrates the process of segregation
of neural and non-neural ectoderm is not completed in ascidians
at the end of gastrulation. Although explantation assays show that
row III/IV cells are specified as neural at the 110 cell stage
(Akanuma and Nishida, 2004; Nishida, 1991), the central cells of
row III/IV are not committed to a neural fate and have been
reported to give rise to the non-neural ectoderm of the oral siphon
(buccal cavity) in Halocynthia rather than contributing to the
neural tube proper (Nishida, 1987) (Fig. 4B). However, this needs
to be confirmed since another study suggested that these cells
contribute to the neural tube in Ciona (Nicol and Meinertzhagen,
1988). Reflecting the uncommitted state of rows III/IV cells in
gastrula stage embryos, they initially coexpress neural transcrip-
tion factors Zic and SoxB1 with non-neural transcription factors
such as DllB, FoxH and Emc, although DllB is then quickly down-
regulated in this domain (Imai et al., 2004; Irvine et al., 2007). It is
currently not known, when and how the adoption of neural fates
in the oral siphon ectoderm is suppressed, but the upregulation of
various non-neural markers such as a second Dlx gene – the Dlx1/
4/6 homologue DllA – as well as Pitx, and Six1/2 in the oral siphon
primordium at early tailbud stages may help to protect it from
commitment to a neural fate (Boorman and Shimeld, 2002;
Caracciolo et al., 2000; Christiaen et al., 2002; Irvine et al., 2007;
Mazet et al., 2005). In striking contrast to amphioxus and verte-
brates, the ascidian oral ectoderm, which will connect with the
pharynx to form the mouth is part of the ectoderm participating in
neurulation and, thus, will become positioned behind the exter-
nally visible “neuropore” and within the primary “neural tube”
(Fig. 4B) (Veeman et al., 2010).
Other metazoans
Taken together these data suggest that parts of the regulatory
network specifying neural vs. non-neural ectoderm in the chor-
date ancestor may have been preserved in ascidians despite
profound changes in upstream inductive processes. Since the
ambulacrarians – the sister group of chordates comprised of
hemichordates and echinoderms – have much more diffuse
nervous systems than chordates and lack an unequivocal homo-
logue of the chordate neural plate (Bullock, 1945; Bullock and
Horridge, 1965; Knight-Jones, 1952; but see below), this network
partitioning the ectoderm into neural and non-neural competence
territories was long considered a chordate innovation. However,
evidence is accumulating that centralised nervous systems
develop from the BMP-depleted side of the dorsoventral axis in
representatives of the ecdysozoans and lophotrochozoans (Arendt
et al., 2008). Moreover, these widely express neural competence
factors of the SoxB1 family (Buescher et al., 2002; Cremazy et al.,
2000; Kerner et al., 2009; Onai et al., 2009; Overton et al., 2002;
Pioro and Stollewerk, 2006). The adjacent non-neural territories
also express some of the chordate non-neural transcription factors.
Dlx, in particular, is widely expressed in the non-neural ectoderm
in many taxa and plays an important role for sensory organ
formation (Cohen and Jürgens, 1989; Denes et al., 2007;
McDougall et al., 2011; Mittmann and Scholtz, 2001; Panganiban
et al., 1997; Plavicki et al., 2012; Prpic and Tautz, 2003; Williams
et al., 2002). AP2 in Drosophila has a similar expression to Dlx but
its expression has not been described in other invertebrates (Bauer
et al., 1998; Monge and Mitchell, 1998). Moreover, the positioning
of expression domains of other transcription factors along the
dorsoventral (medial to lateral: NK2.2, Gsx-NK6-Pax6, and Msx-
Pax3/7) and anteroposterior (rostral to caudal: Otx, Pax2/5/8, and
Hox) axis of the neural ectoderm is also conserved between
insects, annelids and chordates (reviewed in Arendt et al., 2008)
and similar cell types arise from comparable positions along this
molecular coordinate system (De Velasco et al., 2007; Denes et al.,
2007; Tessmar-Raible et al., 2007). Taken together this has led to
suggestions that the establishment of neural and non-neural
territories and the formation of a CNS from the former is
conserved throughout bilaterians and inherited from the last
common bilaterian ancestor (Arendt et al., 2008; Arendt and
Nübler-Jung, 1999; Hirth et al., 2007; Lichtneckert and Reichert,
2005; Urbach and Technau, 2008).
In contrast to chordates, however, Dlx is not expressed in clearly
complementary domains to SoxB1 or other neural transcription
 G. Schlosser et al. / Developmental Biology 389 (2014) 98–119 105

Fig. 4. Anatomy of head region in chordate embryos after completion of neurulation. The neural tube is highlighted in green and the location of the anterior neuropore – a
transitory connection between neural tube and non-neural ectoderm – is indicated by a green asterisk. The non-neural ectoderm is highlighted in orange (general
epidermis), red (oral and preoral part of the epidermis, extending from mouth to anterior neuropore) and pink (palp forming region of epidermis in tunicates). The position
of the developing mouth is indicated by a red asterisk. (A) Amphioxus. Hatschek's left diverticulum (HLD), which forms as an endodermal pouch on the left side, will fuse
with the preoral ectoderm to give rise to Hatschek's pit. (B) Tunicates (Ciona). In tunicates, the oral and preoral parts of the non-neural ectoderm, which form the
primordium of the oral siphon (also known as buccal cavity or stomodeum) participate in neurulation. The resulting externally visible pore of the neurulating ectoderm or
“neuropore” (purple asterisk) is, thus, distinct from the proper neuropore at the terminal end of the neural tube (green asterisk). This connection between anterior neural
tube (sensory vesicle) and oral siphon primordium persists in some larval tunicates with a neurally derived neurohypophysial duct (NHP) and a ciliated funnel and duct
(CFD) derived from the oral siphon primordium. These structures will later form the neural gland of the adult. Most of the larval CNS will degenerate at metamorphosis
except for a few progenitor cells that will give rise to the new cerebral ganglion of the adult. The primordia of the atrial siphons (Atr; hatched brown line) will only develop at
late larval stages by invagination. (B2 and B3) Fatemaps of the so-called “neural plate” of ascidians at 110-cell stage (B2) and mid-gastrula stage (B3) showing the regions of
origin of the neural tube (green), oral and preoral ectoderm (red), palps (pink) and general epidermis (orange) (Nishida, 1987). (C) Vertebrates. Elaboration of the forebrain
results in expansion of the preoral ectoderm, which includes precursors for epidermis and anterior (adenohypophyseal, olfactory, and lens) placodes. The adenohypophyseal
placode buds off the stomodeum as Rathke's pouch (RP) to form the anterior pituitary (A, B1, and C modified from Schlosser, 2005). Abbreviations: Atr, atrial siphon
primordium; CFD, ciliary funnel and duct; Ep, epidermis; HLD, Hatschek's left diverticulum; KP, Kölliker's pit; NHD, neurohypophysial duct; Not, notochord; Nt, neural tube;
OPE, oral-preoral ectoderm; PLP, palps; PP, pharyngeal pouches; RP, Rathke's pouch; SV, sensory vesicle

factors in arthropods and annelids (Cohen, 1990; Denes et al., 2007).
Zic and GATA1/2/3 while being ectodermally expressed in many
protostomes, are often not confined to neural or non-neural ecto-
derm, respectively (Gillis et al., 2007; Layden et al., 2010), while little
is known about other transcription factors such as FoxI. It, thus,
remains unclear whether segregation of neural and non-neural
ectoderm in protostomes is achieved by a similar regulatory network
to chordates. In addition, comparative phylogenetic analyses indicate
that centralised nervous systems that are concentrated on one side of
the dorsoventral axis, are found only in a few scattered animal phyla
106 G. Schlosser et al. / Developmental Biology 389 (2014) 98–119
(in particular arthropods, annelids, and chordates) while most phyla
have either a diffuse nerve net or have evolved a diverse array of
concentrations of neurons in various parts of the body (Bullock and
Horridge, 1965; Holland, 2003; Moroz, 2009). A diffuse nerve net is
also found in enteropneusts (hemichordates) even though many
transcription factors show dorsoventral (e.g. Dlx, PoxNeuro, and
Lim3) or anteroposterior (e.g. Six3/6, Otx, Rx, Pax6, Irx, and Hox)
restrictions in their expression domains similar to chordates (Lowe
et al., 2003, 2006). It has, therefore been alternatively suggested that
centralised nervous systems evolved many times independently,
possibly by convergently using positional information available
through evolutionarily more conserved patterning systems
(Holland, 2003; Lowe et al., 2003, 2006; Moroz, 2009).
At present, this controversy about the shared or independent
origin of bilaterian central nervous systems remains open. Recent
reports suggest that the dorsal and ventral nerve cord of enter-
opneusts are more CNS-like than previously recognised and are
differentiated into cellular layers and neuropils (Brown et al.,
2008; Kaul and Stach, 2010; Nomaksteinsky et al., 2009). In order
to determine whether any of these nerve cords is homologous to
the chordate CNS or rather reflects an independent experiment in
centralisation, it still needs to be analysed whether similar
regulatory networks that distinguish neural from non-neural
ectoderm are employed during development of these nerve cords.
To support the hypothesis of a common bilaterian origin of the
CNS, which would imply multiple independent losses of nervous
system centralisation in several bilaterian phyla, we likewise need
to move beyond comparisons of expression patterns and learn
more about the regulatory networks that partition neural from
non-neural ectoderm in arthropods and annelids.
Origin of specialised neural border territories
Chordates
Whereas the early segregation of ectoderm into neural and non-
neural territories is an ancient chordate trait (and may even date
back to the Urbilateria as discussed in the last section), increasingly
specialised border territories probably evolved within the chordate
lineage (Fig. 3). In amphioxus, a few of the transcription factors that
define the neural plate border in vertebrates are also enriched at the
border of the neural ectoderm such as Pax3/7, Zic and Snail
(Gostling and Shimeld, 2003; Holland et al., 1999; Langeland
et al., 1998; Yu et al., 2008). Msx and Dlx expression, while largely
confined to non-neural ectoderm also extends into lateral neural
ectoderm (Holland et al., 1996; Sharman et al., 1999; Yu et al., 2008).
These genes may promote the formation of dorsal cell types in the
amphioxus neural tube such as the intramedullary sensory neurons
or photoreceptive Joseph cells (Lacalli, 2004; Wicht and Lacalli,
2005) although in the absence of functional studies, this remains
speculation. While a role of these genes for differentiation and
pigment formation of pigmented ocelli (organs of Hesse) has also
been proposed (Yu et al., 2008), this is difficult to reconcile with the
origin of the latter from the medial neural plate.
However, with the exception of Snail, none of the genes
defining the prospective neural crest territory and acting as neural
crest specifier genes in vertebrates are specifically expressed at the
neural plate border in amphioxus. Twist, Ets, FoxD, and Myc are
predominantly expressed in mesoderm (Meulemans and Bronner-
Fraser, 2004; Yasui et al., 1998; Yu et al., 2002b, 2008) and AP2 in
the developing epidermis (Meulemans and Bronner-Fraser, 2002;
Van Otterloo et al., 2012). FoxD, SoxE, Id, Hairy and Irx genes each
have expression domains in the neural plate but none of them is
specifically enriched throughout the border of the neural plate
(Kaltenbach et al., 2009a; Meulemans and Bronner-Fraser, 2004;
Meulemans et al., 2003; Minguillon et al., 2003; Van Otterloo et al.,
2012). Moreover, no migratory or otherwise neural-crest like cells
emerge from the lateral neural plate border in amphioxus.
In ascidians, like in amphioxus, elevated Zic, Pax3/7, Msx and
Snail expression is found at the border of the neural plate as is Ets
(Corbo et al., 1997; Gostling and Shimeld, 2003; Imai et al., 2004;
Ma et al., 1996; Mazet et al., 2003; Squarzoni et al., 2011; Wada
et al., 1996a; Wada and Saiga, 2002; Wagner and Levine, 2012).
Overexpression of Pax3/7 leads to ectopic expression of dorsal
neural marker genes, while Zic knockdown abolishes Snail expres-
sion in the dorsal neural plate implicating both of these genes in
dorsal neural patterning (Imai et al., 2006; Wada et al., 1997). In
contrast to vertebrates, FGF-dependent nodal signalling is crucial
for inducing this lateral neural territory in ascidians (Hudson et al.,
2007; Hudson and Yasuo, 2005). Like in amphioxus, most neural
crest specifiers are not specifically enriched in this territory. SoxE is
not expressed in early development (Imai et al., 2004). AP2 and
Emc, the Ciona Id homologue, are expressed in epidermis, with the
latter being also expressed at the anterior neural plate border. Irx
and Hairy are expressed in parts of the neural plate extending into
its border region but are not specifically restricted to the latter
(Imai et al., 2004, 2009). Twist, FoxD, and Myc are predominantly
expressed in mesoderm/endoderm (Imai et al., 2002,, 2003, 2004;
Tokuoka et al., 2004, 2005). However, FoxD is later also expressed
in the precursor of the ocellus, a lateral neural plate derivative
(Abitua et al., 2012). Since Twist overexpression in these cells
endows them with migratory capacity and their regulatory state
resembles neural crest cells in several ways (expression of Snail,
Ets, Emc, and FoxD; melanogenesis genes) they may be represen-
tatives of a cell population at the lateral neural plate border, from
which the neural crest evolved in the vertebrate lineage (Abitua
et al., 2012). Another population of migratory and HNK positive
cells that give rise to pigmented cells and were initially proposed
to represent neural crest-like cells (Jeffery et al., 2004) are now
known to derive from the mesodermal lineage (A7.6 cells) rather
than from the neural plate border (Jeffery et al., 2008).
In summary, the similarities between amphioxus and Ciona
indicate that at least in the chordate ancestor the lateral neural
plate border was already patterned differently to the medial
neural plate but did not give rise to a neural crest. The latter
probably evolved in the vertebrate lineage by recruitment of new
genes to the lateral neural plate, in particular a suite of genes from
the mesoderm, which promote epithelial–mesenchymal transi-
tions and cell migration (Yu et al., 2008).
Other metazoans
It is currently unclear, whether some of the specialisations of
the lateral neural territory may even predate chordates. Whereas
Msx does not show any dorsoventrally restricted expression in
hemichordates or echinoderms (Dobias et al., 1997; Lowe et al.,
2006), it appears to define lateral domains of the CNS in insects
and annelids and is required for lateral/dorsal specification of
neuroblasts in Drosophila, similar to chordates (D'Alessio and
Frasch, 1996; Denes et al., 2007; Isshiki et al., 1997). Pax3/7 is also
enriched in this lateral domain in annelids (Denes et al., 2007).
Depending on whether the CNS in protostomes is considered
homologous to the chordate neural plate or not (see discussion
above), this suggests either an ancient bilaterian origin of a lateral
neural territory or the convergent recruitment of these genes to a
lateral neural patterning role in protostomes and chordates.
Origin of specialised non-neural border territories
Chordates
In contrast to the neural plate, where clear molecular differ-
ences between dorsal and ventral domains can be recognised, the
 G. Schlosser et al. / Developmental Biology 389 (2014) 98–119 107

non-neural ectoderm of amphioxus appears remarkably uniform
(Fig. 3). None of the genes, which are specifically expressed in the
dorsal non-neural ectoderm of vertebrates such as Six1/2, Six4/5
and Eya are localised to this border region in amphioxus. Rather
these genes are expressed in partly overlapping, partly distinct
domains, for example in the pharyngeal endoderm, the somites,
Hatschek's pit, the anterior neural plate and in primary sensory
cells scattered throughout the non-neural ectoderm (Kozmik et al.,
2007). These sensory cells also resemble placode-derived neurons/
sensory cells of vertebrates because of their migratory capacity,
their probable function as chemo- and/or mechanoreceptors and
the expression of many other markers such as Tlx, Hu/Elav, Islet,
Coe, POU-IV, SoxB1, and Delta (Benito-Gutierrez et al., 2005a;
Candiani et al., 2005, 2006; Holland and Holland, 2001;
Kaltenbach et al., 2009a, 2009b; Mazet et al., 2004; Meulemans
and Bronner-Fraser, 2007; Rasmussen et al., 2007; Satoh et al.,
2001; Schubert et al., 2004). In important distinction to verte-
brates, these sensory cells arise from scattered, but predominantly
ventral precursors in the non-neural ectoderm rather than from a
highly proliferative progenitor population in the dorsalmost non-
neural ectoderm.
Different from amphioxus, ascidians show some regionalisation
of the non-neural ectoderm (Fig. 3) along the dorsoventral axis
with expression of Pax3/7, Msx and some other genes enriched in
its dorsal part (Imai et al., 2004; Mazet et al., 2003; Pasini et al.,
2006; Wada et al., 1996a, 1997). However, the function of Msx in
the epidermis is not clear, while Pax3/7 does not have a dorsalizing
effect on the epidermis (Wada et al., 1997). Similar to the lateral
neural plate, FGF-dependent nodal signalling is crucial for indu-
cing this lateral non-neural territory in ascidians (Pasini et al.,
2006). The dorsal non-neural ectoderm in tunicates also forms
placode-like thickenings around the neural plate and subsequently
invaginates to form the oral siphon primordium anteriorly and the
atrial siphon primordia posteriorly (Bassham and Postlethwait,
2005; Kourakis et al., 2010; Manni et al., 2004b, 2005; Mazet et al.,
2005; Veeman et al., 2010).
Six1/2 and Eya in ascidians and appendicularians are enriched
in dorsal non-neural ectoderm, with Six1/2 in Ciona being
expressed in a horseshoeshaped domain around the anterior
neural plate border resembling the vertebrate pattern (Bassham
and Postlethwait, 2005; Mazet et al., 2005). Also similar to
vertebrates, both DllB and Zic were shown to be required for early
Six1/2 activation in Ciona, although it remains to be determined
whether any of these is required cell-autonomously and with
which signalling molecules they cooperate (Imai et al., 2006). At
later stages, Six1/2 and Eya continue to be expressed in the oral
siphon primordium (and Eya in the adjacent palps as well) and are
also turned on together with Six4/5 in the dorsolateral ectoderm
giving rise to the atrial primordia (Mazet et al., 2005).
Taken together with observations that oral and atrial siphon
primordia and the palp forming region express additional placodal
marker genes and give rise to various primary and secondary
sensory cells (see discussion below) this has led to suggestions
that placodes arising from a common panplacodal primordium are
present in tunicates and, thus, had evolved in the common
ancestor of tunicates and vertebrates after divergence of the
lineage leading to amphioxus (Bassham and Postlethwait, 2005;
Graham and Shimeld, 2013; Manni et al., 2004a, 2004b; Mazet
et al., 2005; Wada et al., 1998). However, it should be noted that in
contrast to vertebrates, the Six1/2 and Eya positive domains of
tunicates do not demarcate regions of massively proliferating
sensory progenitors giving rise to large and complex sensory
organs. Rather only scattered sensory cells or sensory organs
comprising small groups of sensory cells (e.g. the coronal organ
in the oral and cupular organs in the atrial siphon (reviewed in
Holland, 2005; Holland and Holland, 2001)) develop from these
domains. This indicates that proper cranial placodes evolved from
Six1/2 and Eya expressing territories only in the vertebrate lineage
possibly by rewiring of the gene regulatory network downstream
of these genes (Fig. 5; see below for further discussion).
A final point to be addressed here is, whether placodes
originated independently from the neural crest or whether there
is an evolutionary link between the origin of both cell populations.
Even though some genes (e.g. Msx and Pax3/7) are expressed in
both dorsal non-neural ectoderm, and the dorsal neural plate in
tunicates and the border between neural and non-neural ecto-
derm in row III/IV cells is established relatively late (after neural
plate stages), the overall regulatory state of the dorsal non-neural
ectoderm is clearly distinct from the adjacent neural plate but
resembles the adjacent epidermis (Imai et al., 2006). Because
many (but not all) placode-like chemo- and mechanoreceptive
sensory cells in tunicates arise from this Six1/2 and Eya positive

Fig. 5. Scenario for regulatory evolution upstream and downstream of Six1/2 and Eya genes in chordates. It is proposed that the role of Six1/2 and Eya in vertebrate placodes
evolved in 3 steps. Step 1: In the chordate ancestor, Six1/2 and Eya were expressed in sensory cells scattered throughout the non-neural ectoderm regulating their neuronal/
sensory differentiation. This condition is maintained in amphioxus. Step 2: In the common ancestor of tunicates and vertebrates Six1/2 and Eya acquired new upstream
regulators and were recruited to the border of the non-neural ectoderm with the neural plate. However, only scattered sensory cells differentiate from that region, indicating
that Six1/2 and Eya probably cooperate with other factors (X) to promote neuronal/sensory differentiation. This condition is maintained in tunicates. Step3: In the vertebrate
lineage, Six1/2 and Eya acquired new target genes (X and Y) allowing them to adopt additional roles in patterning and proliferation control of neuronal/sensory progenitors.
Neurula stage embryos are shown with the neural plate (NP) indicated in grey. Cells (circles) and regions expressing Six1/2 and Eya are highlighted in red. The position,
where the mouth will form is indicated by a hatched line (modified from Schlosser, 2011).
108 G. Schlosser et al. / Developmental Biology 389 (2014) 98–119
region, the corresponding region in the last common tunicate–
vertebrate ancestor is the most likely precursor region for the
panplacodal primordium in vertebrates. Conversely, the lateral
neural plate with its formation of pigment cells and expression of
ZicL, Snail, and Ets genes is the most likely precursor region for the
vertebrate neural crest. Taken together with many developmental
differences between neural crest and placodes in vertebrates (e.g.
concerning ectodermal competence, time windows of induction,
derivative cell types, expression profile of transcription factors,
mechanisms of morphogenesis) this strongly suggests that
placodes and neural crest have an independent evolutionary
origin, from dorsal non-neural and neural ectoderm, respectively
(Schlosser, 2008).
Other metazoans
In echinoderms and hemichordates, Six1/2 and Eya are pre-
dominantly expressed in endomesoderm including coelomic
pouches and pharyngeal slits (Gillis et al., 2012; Materna et al.,
2013; Yankura et al., 2010). There is no enrichment in dorsal non-
neural ectoderm although there seems to be expression in scat-
tered neuronal or sensory cells. Expression in mechanoreceptors
and rhabdomeric photoreceptors has also been reported from
cnidarians, insects and annelids and functional studies support a
role of Six1/2 and Eya for neuronal differentiation in insect and
planarian eyes (Arendt et al., 2002; Bonini et al., 1993, 1997; Burke
et al., 2006; Cheyette et al., 1994; Graziussi et al., 2012; Halder
et al., 1998; Mannini et al., 2004; Pignoni et al., 1997; Pineda et al.,
2000; Serikaku and O'Tousa, 1994; Stierwald et al., 2004; Suzuki
and Saigo, 2000). Moreover, the neuronal determination gene
Atonal has been shown to be a direct Six1 target in insects as well
as in vertebrates (Ahmed et al., 2012a; Zhang et al., 2006). In the
beetle Tribolium Six1, Six4 and Eya have recently been found to be
expressed in a joint domain at the anterior border of the neural
ectoderm, where they are required for proper development of
epidermis and sensory bristles and it has been proposed that this
insect “head placode” and vertebrate placodes evolved from a
common placode precursor in the bilaterian ancestor (Posnien
et al., 2011a). A domain of Six1/2 expression at the rostral
boundary of the neural ectoderm encompassing the developing
photoreceptors has also been described in annelids (Arendt et al.,
2002) but has not been reported in other protostomes. We
currently lack sufficient comparative information to decide
whether this domain reflects the ancient bilaterian origin of a
Six1/2 territory in the anterodorsal non-neural ectoderm, which
was lost in ambulacrarians and amphioxus, or rather represents
convergent recruitment of Six1/2 to the anterior dorsal non-neural
ectoderm in protostomes and chordates.
However, the association of Six1/2 and Eya expression with
mechanoreceptors and rhabdomeric photoreceptors in cnidarians
and bilaterians supports an ancient role of Six1/2 and Eya in
sensory differentiation (see Schlosser, 2007 for further review). In
deuterostomes, they also have a conserved role in coelomic pouch
and gill slit formation. Thus, similar to extant amphioxus ancestral
chordates probably possessed primary sensory neurons that dif-
ferentiated under control of Six1/2 and Eya and these may be
homologous to some placode-derived neurons (Fig. 5). However,
different from vertebrates these neurons did not arise from foci of
proliferative progenitors. A specialised domain of non-neural
ectoderm expressing Six1/2 and Eya and a few other genes (Pax3/
7 and Msx), was either already inherited from Urbilateria and
subsequently lost in amphioxus or newly evolved in the ancestor
of tunicates and vertebrates. While the transcription factors in this
domain already promoted sensory differentiation and possibly
morphogenetic movements (invagination), additional regulatory
links – e.g. promoting expansion of sensory progenitors and cell
migration – had to be established in the vertebrate lineage for fully
fledged cranial placodes to evolve. We propose that Six1/2
transcription factors while maintaining a role in neuronal differ-
entiation acquired additional roles in promoting patterning and
proliferation of neuronal/sensory progenitors at that stage (Fig. 5)
– a case of intercalary evolution similar to what has been proposed
for Pax6 in eye evolution and for evolution of several other
transcription factors (Davidson, 2006; Gehring and Ikeo, 1999).
Evolution of anteroposterior ectodermal patterning
After considering dorsoventral patterning, we next have to
address, when different placodal territories were established along
the anteroposterior axis in evolution. Again the answer to this
question is complex and will be reviewed here in several steps.
First, we will focus on the emergence of anteroposterior patterning
mechanisms per se and second, we will consider the origin of
different ectodermal territories along the anteroposterior axis, and
the establishment of individual placodes within these domains.
The emergence of anteroposterior patterning mechanisms
Similar to vertebrates, amphioxus uses a Wnt gradient gener-
ated by rostrally secreted Wnt antagonists as well as retinoic acid
(RA) signalling for anteroposterior patterning of both neural and
non-neural ectoderm (Escriva et al., 2002; Holland, 2002, 2005;
Koop et al., 2010; Onai et al., 2009, 2012; Schubert et al., 2004;
Schubert et al., 2006; Yu et al., 2007). However, while some FGFs
are posteriorly confined in amphioxus, they do not seem to be
essential for anteroposterior patterning (Beaster-Jones et al., 2008;
Bertrand et al., 2011). This suggests that the role of at least Wnt
and RA in anteroposterior patterning is not vertebrate specific but
rather represents an ancestral chordate trait. The importance of
these global systems to pattern the anteroposterior axis, has
however been drastically altered in tunicates, due to their evolu-
tion of a peculiar development, which relies heavily on local cell
interactions (reviewed in Lemaire, 2009; Nishida, 2005). As a
consequence, anteroposterior patterning in ascidians is not depen-
dent on a Wnt signalling gradient with exception of the poster-
iormost tail epidermis (Pasini et al., 2012). However, nuclear
translocation of beta-catenin on the vegetal side is crucial for
specifying the animal-vegetative axis and promoting endoderm
formation (Imai et al., 2000). In contrast, retinoic acid has been
implicated in establishing spatially collinear Hox gene expression
and affects other aspects of anteroposterior patterning in asci-
dians, even though its function differs from vertebrates in several
respects and genes for RA synthesis, degradation, and reception
have been completely lost in appendicularians (Cañestro and
Postlethwait, 2007; Kanda et al., 2009; Katsuyama and Saiga,
1998; Katsuyama et al., 1995; Nagatomo and Fujiwara, 2003;
Nagatomo et al., 2003; Pasini et al., 2012). FGFs in ascidians have
also been implicated in posteriorisation of both the developing
CNS and the tail epidermis (Hudson et al., 2003, 2007; Pasini et al.,
2012) suggesting that a role of FGF in anteroposterior patterning
may have evolved in the ancestral lineage of tunicates and
vertebrates.
There is currently no evidence for a role of either FGF or
retinoic acid in anteroposterior patterning outside of chordates.
While FGF signalling is found in all metazoans (Itoh and Ornitz,
2011), it has no consistent role in anteroposterior patterning
outside of chordates. Retinoic acid signalling, in contrast, was long
thought to be a chordate innovation. However, recently a full
complement of RA receptors (RAR and RXR) and enzymes involved
in RA signalling has been found in various lophotrochozoans and
deuterostomes but not in cnidarians or ecdysozoans (Campo-
 G. Schlosser et al. / Developmental Biology 389 (2014) 98–119
Paysaa et al., 2008). This suggests that RA signalling evolved in
bilaterians and was subsequently lost in ecdysozoans but nothing
is currently known about its function outside of chordates.
Unlike FGF and RA, graded Wnt signalling along the antero-
posterior axis and a role for Wnt in conferring posterior identity is
observed in many metazoans. A direct role for Wnt in anteropos-
terior ectodermal patterning has been demonstrated for planar-
ians, some arthropods, for the anteriormost ectoderm in
hemichordates and for cnidarians (Duffy et al., 2010; Marlow
et al., 2013; McGregor et al., 2008; Miyawaki et al., 2004; Pani
et al., 2012; Petersen and Reddien, 2008) even though it is still
debated, whether the Wnt-enriched oral pole of cnidarians corre-
sponds to the anterior (e.g. Martindale and Hejnol, 2009) or
posterior pole of bilaterians (e.g. Holstein et al., 2011; Niehrs,
2010). It has, thus, been suggested that canonical Wnt signalling
has an evolutionary ancient role for patterning the anteroposterior
body axis (reviewed in Holstein et al., 2011; Niehrs, 2010; Petersen
and Reddien, 2009). However, this ancient patterning system has
apparently frequently been modified. In some lineages, Wnt
signalling is not directly involved in positioning transcription
factor domains along the anteroposterior axis but does play earlier
roles in vegetative pole formation and endomesoderm specifica-
tion (e.g. echinoderms, ascidians, and hemichordates) or in pro-
moting vegetal/posterior fate in asymmetric cell divisions
(annelids and nematodes) possibly reflecting secondary modifica-
tions of its primitive role in anteroposterior patterning (Darras
et al., 2011; Huang et al., 2007; Imai et al., 2004; Logan et al., 1999;
Schneider and Bowerman, 2007; Takeshita and Sawa, 2005;
Tokuoka et al., 2004; Wikramanayake et al., 1998, 2003). In other
lineages, Wnt dependent axial patterning is completely lost and
replaced by divergent anteroposterior patterning systems such as
the bicoid gradient in Drosophila (Driever and Nüsslein-Volhard,
1988; McGregor, 2005).
The establishment of ectodermal territories along the anteroposterior
axis
Patterning mechanisms like graded Wnt signalling partition
the ectoderm along the anteroposterior axis by establishing a
sequence of transcription factor territories such as those involved
in placode specification. This raises the question, when the
transcription factor territories which subdivide the preplacodal
ectoderm in vertebrates in response to Wnt and other poster-
iorising signals first arose in evolution.
Chordates
Many of the transcription factors involved in early regionalisa-
tion of the neural and non-neural ectoderm in vertebrates (and
partly other germ layers as well) in response to Wnt and RA
signalling show a similar anteroposterior distribution in
amphioxus with Otx, Fezf (both in neural ectoderm only) and
Six3/6 being anteriorly and Gbx, Cdx, Irx and Hox posteriorly
restricted (Beaster-Jones et al., 2008; Brooke et al., 1998; Castro
et al., 2006; Irimia et al., 2010; Kaltenbach et al., 2009a; Koop et al.,
2010; Kozmik et al., 2007; Pascual-Anaya et al., 2012; Williams and
Holland, 1996) (Fig. 3). Moreover, some of these transcription
factors have been shown to shift their expression boundaries in
response to RA (Hox) or Wnt signalling (Otx and Cdx) (Koop et al.,
2010; Onai et al., 2009). Some additional transcription factors with
regionalised expression in the vertebrate placodal ectoderm also
show anteroposteriorly restricted expression in amphioxus. These
include Pax6, which is expressed in anterior neural tube and
adjacent preoral ectoderm (Glardon et al., 1998; Vopalensky et al.,
2012) and Pitx, which is expressed in preoral ectoderm and
Hatschek's pit (Boorman and Shimeld, 2002; Yasui et al., 2000).
109
However, others are confined to the neural ectoderm (e.g. Pax3/7,
Pax2/5/8, and FoxG1), are expressed in endomesoderm (Pax3/7,
Pax2/5/8, and FoxE4), or have not been analysed in amphioxus
(Dmrt and Anf) (Holland et al., 1999; Kozmik et al., 2007, 1999;
Toresson et al., 1998; Yu et al., 2002a).
There is no evidence in amphioxus for the existence of ectoder-
mal territories homologous to the trigeminal or any posterior (otic,
lateral line, and epibranchial) placodal territories since most tran-
scription factors demarcating these placodes in vertebrates are not
expressed in the non-neural ectoderm of amphioxus. However, a
number of transcription factors expressed in the posterior placodal
area in vertebrates are also coexpressed in the pharyngeal pouches of
vertebrates and amphioxus (e.g. Pax2/5/8, Six1/2, Six4/5, Eya, SoxB1,
Tbx1/10, and Irx) leading to suggestions that a network of genes
originally used in pharyngeal pouch development may have been
recruited for placode specification in vertebrates (Schlosser, 2005).
In contrast, the preoral ectoderm of amphioxus – the anterior-
most part of the non-neural ectoderm located between mouth and
anterior neuropore – expresses a combination of several transcrip-
tion factors (Pax6, Six3/6, and Pitx) found in the extended anterior
placodal area of vertebrates. Id proteins implicated in early
neuronal development and a G-protein coupled receptor related
to vertebrate odorant receptors are also expressed in this domain
(Meulemans et al., 2003; Satoh, 2005). However, in contrast to
vertebrate placodes, no Six1/2, Six4/5 and Eya genes are expressed
and no sensory organs originate from this region, only individual
primary sensory cells, which probably function as chemo- and/or
mechanoreceptors (reviewed in Holland and Holland, 2001;
Lacalli, 2004; Schlosser, 2005). Moreover, similar primary sensory
cells, identified by a number of neuronal differentiation markers
(e.g. POUIV, Coe, and Elav) are found scattered throughout the
epidermis (Benito-Gutierrez et al., 2005a, 2005b; Candiani et al.,
2006; Holland and Holland, 2001; Kaltenbach et al., 2009b; Lacalli,
2004; Mazet et al., 2004; Meulemans and Bronner-Fraser, 2007;
Satoh et al., 2001). Thus apart from a potential enrichment of
olfactory-type chemosensory cells, there is little evidence for
placode-like specialisations in this domain in amphioxus.
There is, however, Hatschek's pit, which in addition to Pax6,
Six3/6 and Pitx expresses a number of additional transcription
factors found in the adenohypophyseal placode of vertebrates such
as Pit1, Lhx, Islet as well as Six1/2 and Eya (Candiani et al., 2008;
Jackman et al., 2000; Kozmik et al., 2007; Wang et al., 2002). As
discussed in the first part of this review (Patthey et al., in this
issue) there is also evidence that it acts as a neurosecretory organ,
although adenohypophyseal hormones only evolved in the verte-
brate lineage.
The embryonic origin of Hatschek's pit also differs markedly
from the adenohypophysis (Fig. 4A). Hatschek's pit arises from a
fusion of Hatschek's left diverticulum with the preoral ectoderm
(Holland and Holland, 2001). The former originates as a pouch
from the anterior gut and is, thus, of endomesodermal proveni-
ence, whereas the adenohypophysis of vertebrates is purely
ectodermal and arises by invagination of the adenohypophyseal
placode. This ectodermal origin has now been confirmed also for
the hagfish adenohypophysis (Oisi et al., 2013), for which a
previous report had suggested an endomesodermal origin
(Gorbman, 1983). However, connections between the adenohypo-
physis and adjacent endomesodermal head cavities have been
observed in some vertebrates, resembling the situation in
amphioxus (Goodrich, 1917), and it has been suggested that
expression domains of transcription factors in the endomesoder-
mal part of Hatschek's pit may have expanded and shifted to the
adjacent ectoderm during chordate evolution (Schlosser, 2005).
Taken together, this suggests that the anteriormost placodes of
vertebrates may have evolved from a region of preoral ectoderm
giving rise to a neurosecretory organ like amphioxus' Hatschek's
110 G. Schlosser et al. / Developmental Biology 389 (2014) 98–119
pit. It has been previously proposed that by recruitment of
olfactory receptor neurons and GnRH releasing cells this structure
may have evolved into a first rostral placode, from which other
vertebrate placodes subsequently diversified (Schlosser, 2005).
As discussed above, tunicates rely much less on patterning
gradients than other chordates. Presumably as a consequence,
some transcription factors important for early anteroposterior
regionalisation in other chordates have been lost from the genome
(e.g. Gbx) or changed their expression pattern (e.g. Cdx) (Ikuta
et al., 2010; Wada et al., 2003). The Hox cluster has been disrupted
with loss of temporal colinearity and reduced anteroposterior
patterning functions (Ikuta et al., 2010; Ikuta et al., 2004; Seo
et al., 2004). Nevertheless, the expression domains of several other
transcription factors along the anteroposterior axis resemble the
situation in vertebrates and amphioxus (Fig. 3), indicating that
they have been evolutionarily preserved despite changes in the
upstream patterning mechanisms. Anterior ectoderm (including at
least the a-line neural plate giving rise to anterior neural tube, oral
siphon primordium and palps; Fig. 4B), is characterised by Otx
expression with Emx and Six3/6 (Fezf has not been described)
being expressed in anterior (prospective palps) and posterior
(prospective oral siphon primordium and anterior sensory vesicle)
part of this domain, respectively (Imai et al., 2004; Mazet et al.,
2005; Moret et al., 2005; Oda and Saiga, 2001; Wada et al., 1996b).
Posterior ectoderm, in contrast, is marked by expression of Irx and
Hox transcription factors (Ikuta et al., 2004; Imai et al., 2004;
Pasini et al., 2012).
In addition, several transcription factors involved in delimit-
ing the extended anterior placodal area (giving rise to adenohy-
pophyseal, olfactory and lens placodes) of vertebrates are
expressed in restricted domains of the ascidian non-neural
border. These include Pitx in the oral siphon primordia and FoxG
with high expression in the developing palps, whereas others are
confined to the neural tube (Pax6), the endomesoderm (FoxE) or
have not been analysed (Anf) (Boorman and Shimeld, 2002;
Christiaen et al., 2002; Glardon et al., 1997; Imai et al., 2004;
Mazet et al., 2005; Ogasawara and Satou, 2003; Tiozzo et al.,
2005). Dmrt is also expressed on both neural and non-neural
sides of the anterior neural plate border, where it promotes FoxC
in the palp territory in the absence of FGF and acts as upstream
regulator of Six1/2 (Imai et al., 2006; Wagner and Levine, 2012).
More posterior, Pax2/5/8, one of the defining markers of the
vertebrate posterior placodal area (giving rise to otic, lateral line
and epibranchial placodes), is not only expressed in the “neck”
region of the neural tube, but also in the non-neural ectoderm of
the atrial siphon primordia, which invaginate at late larval stages
to form the atrium around the adult gill basket; it is, however,
also expressed in the oral siphon primordium (Mazet et al., 2003;
Wada et al., 1997, 1998).
As discussed in the first part of this review (Patthey et al., in
this issue), due to its position and similarities in gene expression
(Pitx, Six1/2, Eya, and Six3/6) the oral siphon primordium, in
particular its posterior wall, which remains connected with the
anterior neural tube of the larva (sensory vesicle) by the neuro-
hypophyseal duct, has been proposed to be homologous to the
adenohypophyseal placode (Boorman and Shimeld, 2002;
Christiaen et al., 2002; Graham and Shimeld, 2013). In adult
ascidians, the neurohypophyseal duct persists as the neural gland,
which opens into the oral siphon via a ciliated funnel and duct
(derived from the posterior wall of the oral siphon primordium)
whereas the larval neural tube (sensory vesicle and visceral
ganglion) degenerates and a new cerebral ganglion forms by cells
migrating out of the larval neural tube (Horie et al., 2011; Manni
et al., 1999, 2004b). Although precise fate mapping studies have
not been performed, the oral siphon primordium probably also
gives rise to several types of sensory cells in tunicates. These
include the presumptive photoreceptors of the oral siphon pig-
ment organs, small groups of primary sensory cells, and the
secondary sensory cells of the coronal organ, which encircles the
oral tentacles (Auger et al., 2010; Bassham and Postlethwait, 2005;
Burighel et al., 2003; Caicci et al., 2013; Manni et al., 2004a, 2004b,
2006). Neurosecretory cells (e.g. GnRH positive cells) are known to
arise from the adjacent sensory vesicle, but there is currently no
evidence for such cells to originate from the oral siphon primor-
dium itself (Hamada et al., 2011; Kavanaugh et al., 2005; Kusakabe
et al., 2003).
Sensory neurons are also concentrated in the palps of ascidians
or in comparable regions in other tunicates, which develop in the
non-neural ectoderm derived from row V/VI of the a-line “neural
plate” immediately rostral to the oral siphon primordium. These
express neuronal differentiation markers COE, and POUIV in
addition to Eya (Bassham and Postlethwait, 2005; Candiani et al.,
2005; Mazet et al., 2004; Takamura, 1998) and were suggested to
be olfactory placode homologues (Mazet et al., 2005).
Finally, the primordia of the atrial siphons were suggested to be
homologous to otic placodes based on their position, invagination,
induction by FGF, gene expression (Pax2/5/8, FoxI, Six1/2, Six4/5,
Eya, and some neurogenic markers) and the origin of sensory
organs (e.g. cupular and capsular organ, Bone and Ryan, 1978;
Mackie and Singla, 2003, 2004) from these primordia (Graham and
Shimeld, 2013; Jefferies, 1986; Kourakis et al., 2010; Kourakis and
Smith, 2007; Mackie and Singla, 2004; Manni et al., 2004b; Mazet
et al., 2005; Wada et al., 1998). Many of the genes expressed in
atrial pimordia are also associated with gill slit formation in
deuterostomes raising the possibility that their original function
was in gill pore formation and that they were subsequently
recruited to adopt other functions in the posterior cranial ecto-
derm overlying the pharyngeal region (Gillis et al., 2012; Holland,
2005; Kozmik et al., 2007; Schlosser, 2005).
However, how the sensory cells of the atria relate to the
sensory cells derived from the otic and lateral line placodes is still
controversially discussed (see Patthey et al., in this issue) because
atrial sensory organs are composed of primary (axon-bearing) and
not secondary (axon-less) sensory cells, in contrast to the sensory
organs of the vertebrate inner ear and lateral line. Since secondary
sensory cells in tunicates are confined to the coronal organ, others
have proposed the latter to be homologous to the vertebrate otic
and lateral line placodes (Manni et al., 2006). However, given the
rostral origin of the coronal organ, the ectodermal domain from
which it develops is more likely positionally homologous to the
vertebrate rostral placodes, despite producing secondary sensory
cells. As discussed before (Patthey et al., in this issue), the
secondary sensory cells of the coronal organ may instead be
merely cellular homologues of vertebrate hair cells, which initially
may have evolved in a rostral proto-placodal domain in the
tunicate–vertebrate ancestor, but were then recruited for the
evolution of otic/lateral line placodes in the vertebrate lineage.
Alternatively, the secondary sensory cells of the vertebrate otic/
lateral line placode may have evolved convergently from a more
caudal population of primary sensory cells as found in the atrial
primordia (this may have happened either in the vertebrate
lineage or already in the ancestors of tunicates and vertebrates
with subsequent loss of secondary sensory cells in tunicates).
In summary, current evidence shows that tunicate palps/oral
siphon primordia and atrial siphon primordia share some expres-
sion domains of anteroposteriorly regionalised transcription fac-
tors with the extended anterior (adenohypophyseal/olfactory/lens)
and posterior (otic/lateral line/epibranchial) placodal areas of
vertebrates, respectively. This suggests that oral and atrial tunicate
siphon primordia and anterior and posterior groups of placodes in
vertebrates evolved from anterior and posterior proto-placodal
ectodermal domains in the tunicate–vertebrate ancestor,
 G. Schlosser et al. / Developmental Biology 389 (2014) 98–119
respectively. This is also supported by a recent phylostratigraphic
study, which suggested that evolutionary origin events for olfac-
tory, otic and lateral line specific genes are enriched in the
tunicate–vertebrate stem lineage (Sestak et al., 2013). Note that
we use the term “proto-placodal” here in a wider sense than in
(Schlosser, 2005) – where it was used to describe the first cranial
placode – to denote regions of the non-neural ectoderm that even
though they are not proper cranial placodes are positionally
homologous to placode-forming ectodermal territories in verte-
brates and which may have undergone some morphogenetic
movements and may have formed some sensory cells. Given the
presence of rostral neurosecretory cells in amphioxus, neurose-
cretory cells may also have been present in the rostral proto-
placodal ectodermal domain of the tunicate–vertebrate ancestor
being subsequently lost in tunicates. In addition, some sensory cell
types originating from these proto-placodal regions may be
homologous to sensory cells found in the olfactory and otic/lateral
line placodes although this requires more study. However, proper
cranial placodes, which give rise to high density arrays of specia-
lised sensory receptors, sensory neurons and supporting cells
evolved from these domains only in the vertebrate lineage. Since
these features are shared between different placodes arising from
the preplacodal ectoderm in vertebrates, their origin most likely
reflects the rewiring of the gene regulatory network downstream
of genes defining the preplacodal region such as Six1/2, Six4/5 and
Eya in the vertebrate lineage (see discussion above).
Other metazoans
Several of the early transcription factor domains set up along
the anteroposterior axis in response to graded Wnt signalling in
chordates probably have a role in anteroposterior patterning that
predates the chordates. Staggered anteriorly restricted expression
of Six3, Fezf, Otx, Emx and posteriorly restricted expression of Irx,
Gbx, and Hox has also been observed in hemichordates (with
divergent patterns for Fezf and Irx), insects and annelids, and thus
in all major clades (deuterostomes, ecdysozoans and lophotro-
chozoans) of bilaterians (Aronowicz and Lowe, 2006; Hirth et al.,
2003; Irimia et al., 2010; Lowe et al., 2003; Pani et al., 2012;
Posnien et al., 2011b; Santagata et al., 2012; Sen et al., 2013;
Steinmetz et al., 2011, 2010; Tomer et al., 2010). Moreover, Otx and
Gbx form abutting domains in all of these lineages and Fezf–Irx are
abutting in insects (Hirth et al., 2003; Irimia et al., 2010; Steinmetz
et al., 2011). In addition, FoxG1 is associated with the rostralmost
neural ectoderm in all lineages, while Pax2/5/8 was shown to be
expressed at the border of Otx and Gbx domains in the insect brain
and hemichordate ectoderm (Grossniklaus et al., 1992; Hirth et al.,
2003; Lowe et al., 2003; Pani et al., 2012; Tomer et al., 2010). The
conserved expression of these early transcription factors along the
anteroposterior axis has first been noticed for the neural ectoderm
and has led to suggestions that all bilaterians inherited a “tripar-
tite” organisation of the brain from urbilaterians (Hirth et al.,
2003; Lichtneckert and Reichert, 2005, 2008). However, many of
them show regionalised expression in non-neural ectoderm as
well suggesting that they have more general roles in early
regionalisation of ectoderm.
In cnidarians, Six3 is aborally confined and some Hox proteins
show restricted expression but expression of other transcription
factors involved in early anteroposterior patterning including Otx
and the direct Wnt targets Gbx, Irx, and Cdx differs from
bilaterians or has not been analysed (e.g. Fezf and FoxG) (Chiori
et al., 2009; Matus et al., 2006; Mazza et al., 2007; Ryan et al.,
2007; Sinigaglia et al., 2013). This suggests that while a role of
graded Wnt signalling in anteroposterior patterning and the role
of some transcription factors in regionalizing the anteroposterior
axis may predate bilaterians, other transcription factors (e.g. Otx,
111
Gbx, Irx, and Cdx) adopted a role in early anteroposterior pattern-
ing only in bilaterians.
Many of the transcription factors involved in further subdivid-
ing the non-neural ectoderm in chordates, such as Pax, Dmrt and
Pitx proteins probably adopted their role in anteroposterior
patterning more recently from ancient functions in cytodifferen-
tiation (Pax and Dmrt) or left-right patterning (Pitx). For example,
PaxB, which gave rise to the Pax2/5/8 and Pax4/6 families, is not
expressed in an anteroposteriorly restricted manner in cnidarians,
but plays an important role in sensory differentiation of photo-
and mechanoreceptors (Fritzsch et al., 2005; Kozmik et al., 2003;
Matus et al., 2007; Piatigorsky and Kozmik, 2004). Both Pax6 and
Pax2/5/8 subsequently acquired regionalised expression in the
neural (and sometimes non-neural) ectoderm although the
homology of many expression domains between taxa remains
controversial (Arendt et al., 2002; Backfisch et al., 2013; Denes
et al., 2007; Lowe et al., 2003; Urbach and Technau, 2003). Other
transcription factors like Dmrt (as well as Six1/2 discussed above)
appear to also have had an original function in cytodifferentiation
(e.g. Parlier et al., 2013) and evolved a role in anteroposterior
patterning only later.
In contrast, Pitx plays a central role in left-right patterning in
deuterostomes and also controls chirality of shell coiling in snails
suggesting that it may have an ancient bilaterian role in left-right
patterning (Grande and Patel, 2009a; Grande and Patel, 2009b). In
hemichordates it is expressed in the proboscis pore (Lowe et al.,
2006), which forms asymmetrically (either on the left or on
the right side) (Benito and Pardos, 1997). Interestingly, while
the proboscis pore is on the dorsal side in hemichordates, its
putative homologue in amphioxus, Hatschek's pit (Goodrich, 1917),
is positioned ventrally immediately rostral to the mouth. This
suggests that Pitx acquired its new role in patterning anterior non-
neural ectoderm (regulated by independent enhancers; Christiaen
et al., 2005; Yoshida and Saiga, 2008) only after dorsoventral axis
inversion and formation of a new mouth in chordates.
Summary and reappraisal of placode evolutionary origins
In conclusion, it is becoming increasingly clear that patterns of
transcription factor expression may be shared between sense
organ primordia of different phyla even when these sense organs
have evolved independently and thus are not homologous for at
least two (not mutually exclusive) reasons: (1) sense organs may
form at similar regions of the body (and for functional reasons
will be concentrated anteriorly) with positioning being controlled
by evolutionarily ancient patterning mechanisms, homologous
between phyla; (2) sense organs may be composed of homologous
cell types, controlled by transcription factors with an ancient role
in cytodifferentiation (which may or may not have additional
patterning roles as well). Consequently, for example olfactory and
mechanosensory organs in vertebrates, which are probably novel
placode-derived structures that evolved in the vertebrate lineage,
share expression of some cytodifferentiation and patterning genes
with (most likely independently evolved) olfactory and mechan-
osensory organs in other phyla (Boekhoff-Falk, 2005; Fritzsch
et al., 2005, 2007; Sen et al., 2013). The independent evolution
of complex eyes from rostrally concentrated ciliary and rhabdo-
meric photoreceptors and the evolution of neuroendocrine brain
centres from rostrally enriched neurosecretory cells provide
further well studied examples for this process (Arendt, 2003;
Arendt et al., 2004; Steinmetz et al., 2010; Tessmar-Raible et al.,
2007; Vopalensky and Kozmik, 2009).
Some of the previous hypotheses of placode evolution dis-
cussed in the first part of our review (Patthey et al., this issue)
appear as too simplistic or untenable in the light of recent data.
112 G. Schlosser et al. / Developmental Biology 389 (2014) 98–119

Fig. 6. Key steps in placode evolution. Evolutionary changes of ectodermal patterning mechanisms and origin of new cell types are mapped onto the phylogenetic tree of
metazoans. Important nodes are numbered and the key depicts which characters can be traced to each node. Character origination events whose placement on the tree is
controversial are indicated by a question mark. AP: anteroposterior; DV: dorsoventral; TF: transcription factor.

However many contain a degree of truth, in that they highlight
when certain placode characters may have evolved (or, more
accurately, what is the most ancient part of the tree of life that
they can convincingly be traced to). In this sense some disagree-
ments in the literature over placode evolution might be reconciled
were agreement to be reached on the level of homology or type of
character under consideration.
With this in mind, in Fig. 6 we attempt to summarise what
currently can be inferred about the evolutionary history of
placodal cell types and patterning mechanisms. It is clear that
sensory and neurosecretory cell types as well as many aspects of
anteroposterior and dorsoventral ectodermal patterning predate
vertebrate origins by a considerable period, sometimes being as
old or even older than bilaterians. However, these cell types and
 G. Schlosser et al. / Developmental Biology 389 (2014) 98–119
patterning mechanisms have been modified and elaborated during
evolution and have been recombined and redeployed in new ways
to give rise to novel structures such as the cranial placodes in the
vertebrate lineage.
Future prospects
While much progress has been made in recent years, there are
still significant gaps in our knowledge of placode evolution. First,
we still lack sufficient grasp of protochordate embryology.
Tunicates are well described up to the early larval stage, but
beyond this cell fates are not well known. For amphioxus, we have
excellent tissue level descriptions but lack cell level descriptions.
In particular, lineage tracing through early development in
amphioxus, and through larval development and metamorphosis
in tunicates, would address some remaining questions.
Second, we have insufficient information on the gene regula-
tory networks controlling ectodermal patterning in all chordates,
and especially in amphioxus. Dissection of these will allow
comparison between the different chordate groups, allowing a
much deeper understanding of the interface between ancient
mechanisms that are homologous between different chordates,
and vertebrate novelties. This may also give us insight into how
evolutionary innovations have happened by rewiring of gene
networks, for example by identifying new regulatory interactions,
or new associations between preexisting regulatory modules.
Finally, we currently have little insight into the evolutionary
history of placode-derived cell-types as discussed in the first part
of this review (Patthey et al., in this issue). Comparative transcrip-
tomic approaches using high-throughput sequencing are likely to
help us answer these questions. Transcriptomics offers the oppor-
tunity to ask how placode cell types relate to each other, how they
relate to similar cells that form elsewhere in the vertebrate
embryo, and how they relate to similar cells in tunicates,
amphioxus and indeed other animals. While much has been
learned, there is still much to discover and likely many more
surprises along the way.
Acknowledgements
We would like to express our gratitude to Fondation Les Treilles
for their support for this meeting, and Marianne Bronner, Carole
LaBonne and the other participants for helpful discussions and
insights. Research on placode evolution in the Schlosser lab is
supported by Grant 11/RFP/EOB/3168 (Science Foundation Ire-
land). CP and SMS would like to thank The Royal Society and
EMBO for support.
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