Gene regulatory network for neurogenesis in
a sea star embryo connects broad neural
specification and localized patterning
Kristen A. Yankura1, Claire S. Koechlein2, Abigail F. Cryan, Alys Cheatle, and Veronica F. Hinman3
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213
Edited by Eric H. Davidson, California Institute of Technology, Pasadena, CA, and approved April 17, 2013 (received for review December 3, 2012)
A great challenge in development biology is to understand how
interacting networks of regulatory genes can direct the often
highly complex patterning of cells in a 3D embryo. Here, we detail
the gene regulatory network that describes the distribution of
ciliary band-associated neurons in the bipinnaria larva of the sea
star. This larva, typically for the ancestral deuterostome dipleurula
larval type that it represents, forms two loops of ciliary bands that
extend across much of the anterior-posterior and dorsal-ventral
ectoderm. We show that the sea star first likely uses maternally
inherited factors and the Wnt and Delta pathways to distinguish
neurogenic ectoderm from endomesoderm. The broad neurogenic
potential of the ectoderm persists throughout much of gastrulation.
Nodal, bone morphogenetic protein 2/4 (Bmp2/4), and Six3-dependent
pathways then sculpt a complex ciliary band territory that is de-
fined by the expression of the forkhead transcription factor,
foxg. Foxg is needed to define two molecularly distinct ectodermal
domains, and for the formation of differentiated neurons along the
edge of these two territories. Thus, significantly, Bmp2/4 signaling in
sea stars does not distinguish differentiated neurons from nonneuro-
nal ectoderm as it does in many other animals, but instead contrib-
utes to the patterning of an ectodermal territory, which then, in turn,
provides cues to permit the final steps of neuronal differentiation.
The modularity between specification and patterning likely reflects
the evolutionary history of this gene regulatory network, in which
an ancient module for specification of a broad neurogenic potential
ectoderm was subsequently overlaid with a module for patterning.
encoded within the genome and initiated by anisotropies in
the zygote, can orchestrate the complex final pattern of appro-
priately differentiated cells in time and 3D space. The specification
and patterning of the nervous system has received particular
attention from developmental biologists, as it is such a defining
feature of the body plan. Many animals have diffuse nervous
systems that form a neural net (1). Other animals have highly
patterned centralized nervous systems (CNSs), of which the best
characterized are the ventral nerve cord of Drosophila and the
dorsal hollow neural tube of vertebrates. In both of these very
disparate taxa, the position of the neuroectoderm is established
by gradients of bone morphogenetic protein (BMP) and its an-
tagonist, Chordin/short gastrulation (Sog), along the dorsal-
ventral (DV) axis (2, 3). High concentrations of BMP promote
the formation of nonneurogenic ectoderm, whereas neurogenic
ectoderm forms where BMP concentration is low (4). Although
there are other types of nervous system localizations (1), much
less is known of the GRNs that lead to their final pattern. We
were especially intrigued by the particularly distinctive local-
ized pattern of neurons associated with the ciliary bands of the
bipinnaria larva of the sea star, Patiria miniata. The sea star
(Phylum Echinodermata) bipinnaria has two loops of ciliary
bands: one that loops above the mouth and one below it, which
extends from the ventral surface to the anterior, dorsal margins
he fascinating challenge of developmental biology is to de-
termine how gene regulatory networks (GRNs), which are
of the ectoderm (Fig. 1A). As there are two loops of ciliary
bands, a particular anterior-posterior (AP) coordinate may
have neurons along two locations within the DV axis and vice
versa. These neurons coordinate the action of the cilia to enable
the larvae to swim and feed in response to environmental cues
within the water column (5). This design is very different from
the DV-restricted patterning of the CNS in vertebrates and
Drosophila, and even the echinoderm pluteus larva, which develops
neurons associated with a single ciliary band that is restricted along
the DV axis (Fig. 1A). Indeed, in sea stars there seems to be no
simple AP and DV positioning mechanism that could establish the
pattern of these neurons. However, unlike other well-characterized
deuterostome embryos, this distinctive bipinnaria pattern of neu-
rons is a common feature of the larvae of members of several
classes of echinoderms [i.e., the auricularia of sea cucumbers (6)
and the larvae of some basal crinoids (7)] and also some groups of
hemichordates (i.e., tornaria larvae). This larval type has therefore
arguably been defined as the ancestral deuterostome form and, as
such, has featured strongly in evolutionary hypotheses of vertebrate
CNS origins (8–10). However, very little is known of the mecha-
nisms of development of this larva, particularly of the ectoderm.
Thus, the motivation of this study was to understand the GRN that
explains a distinctive nervous system pattern in what might be the
basal representative of a large clade of animals.
The initial placeholders of the GRN for this developmental
process are the signaling events that first establish the AP and
DV axes and distinguish ectoderm from endoderm and mesoderm.
Sea star embryos undergo equal cleavage and hatch as a blastula
at around 24 h after fertilization. The endoderm and mesoderm
form at the vegetal pole and invaginate during gastrulation, leaving
the ectoderm as a ciliated outer domain. Recent studies indicate
that canonical Wnt (cWnt) pathways may have an ancestral role
in establishing early animal (anterior)-vegetal (posterior) axes and
distinguishing endomesoderm from ectoderm (11, 12). We there-
fore start our examination of the AP axis formation and the es-
tablishment of the ectoderm by considering the role of cWnt
signaling in the sea star. The first morphological evidence of DV
patterning in sea stars is the formation of an invagination (where
Author contributions: K.A.Y. and V.F.H. designed research; K.A.Y., C.S.K., A.F.C., and A.C.
performed research; K.A.Y. and V.F.H. analyzed data; and K.A.Y. and V.F.H. wrote
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The Patiria miniata cDNA sequences reported in this paper have been
deposited in the GenBank database [accession nos.: JX844799 (ephrin), JX844800 (ephrin
receptor), JX844801 (elav), JX844802 (soxb1), JX844804 (soxc), JX844803 (wnt8), KC669537
(nodal), and KC669538 (bmp2/4)].
1Present address: Department of Microbiology, Immunology, and Molecular Genetics,
University of California, Los Angeles, CA 90095.
2Present address: Department of Pharmacology, University of California San Diego School
of Medicine, La Jolla, CA 92093.
3To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| May 21, 2013
| vol. 110
| no. 21
the mouth will later from) on the ventral surface at around 72 h.
This formation is preceded by the expression of the transcription
factor foxa in this oral territory before gastrulation (13). In sea
urchins, Nodal is needed to establish the mouth ectoderm and BMP
gradients are used to establish territories along the DV ectodermal
axis (14–17). These pathways therefore also serve as starting points
for our investigations of DV patterning in the sea star.
Results and Discussion
Localized Patterns of Neurons in the Sea Star Larva. We first char-
acterized the pattern of neurons by examining the expression of
the single P. miniata ortholog of elav (Fig. S1). Elav is an RNA
binding protein that plays a role in the transition from neural
progenitor to committed and differentiated states (18) and is a
conserved molecular marker of postmitotic neurons (19, 20). Ex-
pression is detected in two rows of ectodermal cells along the
pre- and postoral ciliary bands that surround the mouth (Fig. 1 B
and C and Fig. S2A); additionally, elav expression is present in
the lower lip of the mouth and esophagus (Fig. S2B) and within
the bilateral anterior dorsal ganglia (Fig. S2C). Earlier in deve-
lopment, elav is first observed within several cells in the anterior
pole ectoderm of 2-d-old (gastrula stage) embryos (Fig. 1D). We
think that these elav-positive cells will later coalesce to form the
bilateral, anterior dorsal ganglia of larvae, rather than neurons of
the ciliary bands. Previous studies have characterized the distri-
bution of neurons in the sea star bipinnaria based on immuno-
reactivity to the serotonin precursor, 5-hydroxytryptophan, and
synaptotagmin-B (21). These studies identify neurons associated
with the anterior ectoderm, larval ciliary bands, lower lip of the
mouth, and esophagus (22, 23). Thus, we are confident that elav
expression within the ectoderm serves as a marker of most, if
not all, differentiated neurons. Expression of elav is observed
additionally within the mesodermal bulb at the top of the arch-
enteron of gastrulae (Fig. 1D), and later in these cells as they
ingress into the blastocoel. It is not yet known if any of these elav-
expressing mesenchymal cells are neurons.
This finding is very different from the pattern of neurons found
in the two closest relatives to the sea star for which any mech-
anisms of neural specification are known (i.e., the sea urchin and
the directly developing hemichordate Saccoglossus, which con-
trasts to the indirectly developing hemichordates that develop
tornaria larvae). In sea urchins, a single ciliary band and its
associated neurons (22) form at the boundary between the oral
(ventral) and aboral (dorsal) ectoderm (14–17, 24) (Fig. 1F). In
Saccoglossus, elav is expressed throughout the ectoderm with no
apparent DV patterning (20).
Neurogenesis Occurs Broadly Throughout the Ectoderm During
Gastrulation. We previously identified many transcription factors
expressed within the sea star ectoderm (25–27) that, based on
their orthology to other taxa, likely have roles in neurogenesis.
P. miniata orthologs of otx (in particular the ectodermally lo-
calized otxßb isoform) (26), onecut (see ref. 27 and Fig. 2A), and
soxb1 (Fig. 2B) are expressed throughout the ectoderm through
gastrulation. Quantitative reverse-transcription PCR (qPCR)
reveals that transcripts of sea star orthologs of these genes, as well
star embryos are oriented with the anterior pole up and with ventral to the
left: h, hours postfertilization; LV, lateral view; VV, ventral view. (A) Expression
of onecut within the ectoderm marks the ciliary bands that loop above and
below the mouth (Mo) of 96-h-old larvae (WMISH). (B) Dotted lines highlight
elav-expressing ectodermal cells in two rows above and below the mouth.
elav-expressing cells are also detected within the mesoderm and mesenchyme
(not in plane of focus in B) (WMISH). (C) Two rows of elav-expressing cells
(green) form near the ciliary bands, marked by foxg (purple) (FISH). (D) Tran-
scripts of elav are first observed within cells of the anterior ectoderm (arrows)
and in the mesodermal bulb of the archenteron (AR) of gastrulae (WMISH).
Schematic of sea star (E) and sea urchin (F) larvae, viewed laterally, depicts
expression of elav (magenta) in all known neural territories; the larval ciliary
bands (CB) are shown in light purple. DG, dorsal ganglion; ES, esophagus;
LL, lower lip. (Magnification: 200×.)
Expression of elav in P. miniata. In all figure plates, unless noted, sea
Transcripts of onecut (A), soxb1 (B), and soxc (C) are found throughout the
ectoderm of gastrulae. (D) soxc-expressing cells (purple) do not colocalize
with those expressing glial cells missing (gcm; green) (32). (E) Ectopic ex-
pression of soxc within the ectoderm of Delta morphants (MO). (F) Tran-
scripts of elav in the ciliary band neurons of an ∼72-h-old larva. In Onecut
morphants at ∼72 h (G) or 96 h (H) postfertilization, elav-positive ciliary band
neurons are not observed. (I) Both anterior and ciliary band elav-expressing
neurons are lost in Soxc morphants around 72 h postfertilization. elav remains
expressed in the mesoderm (G–I) at the tip of the archenteron (AR), later in
mesenchyme (not in plane of focus in H and I) and in some anterior ectodermal
cells (arrows in G and H). (Magnification: 200×.)
Sea star gastrulae develop a broad neurogenic potential ectoderm.
| www.pnas.org/cgi/doi/10.1073/pnas.1220903110 Yankura et al.
as other pan-ectodermal–expressed regulatory genes (25), are
abundant within the fertilized eggs (Figs. S3 and S4), and there-
fore may operate near the top of a developmental GRN hier-
archy for specification of the ectoderm. When cWnt signaling is
blocked by injecting one-cell–stage embryos with a construct that
blocks the nuclearization of β-catenin (Δ-cadherin) (28), the
expression of these genes is found throughout the later embryo
(Fig. S5 A and B). elav-expressing cells are also found scattered
throughout this nβ-catenin–deficient ectoderm (Fig. S5C) rather
than being restricted the anterior pole neural territory (e.g., com-
pare with Fig. 1D). Thus, we suggest that similar to sea urchins
and many other taxa, cWnt signaling is used to initially distin-
guish ectoderm from endomesoderm. In particular, cWnt sig-
naling in sea stars segregates—as we show further below—the
broad neurogenic potential ectoderm from the endomesoderm.
In vertebrates, and possibly protostomes (29), the expression
of sox gene-family members marks a progression along a pathway
toward neural commitment; soxb orthologs function during the
early specification of neurons, and the soxc orthologs play a role
in the commitment of neurons (30). We find here that in sea stars,
soxb1 is expressed throughout the ectoderm of gastrulae (Fig. 2B),
but transcripts of soxc are also found throughout the ectoderm,
but only within distinct cells, giving the appearance of a spotted
pattern of localization (Fig. 2 C and D). Delta Notch lateral in-
hibition is a conserved mechanism for distinguishing one cell type
from another, particularly neuronal from nonneuronal, within an
epithelium (31). In sea stars, delta is expressed in a spotted pat-
tern throughout the ectoderm at the gastrulae stage (32) and,
when disrupted, results in an expansion of soxc expression to
adjacent cells within the ectoderm (compare Fig. C and E). This
finding suggests that a lateral inhibition mechanism may also be
used to segregate soxc-expressing cells within the ectoderm.
We next sought to determine whether the expression of elav
depends on these pan-ectodermal–expressed genes. Perturbation,
by injecting zygotes with a sequence-specific translation blocking
morpholinos (hereafter referred to as a morphant) of onecut
results in the loss of elav-expressing cells associated with the
developing ciliary bands (compare Fig. 2 F and G). Some elav-
expressing cells remain within the anterior pole territory in what
we think are cells that will later become the dorsal ganglia (Fig.
2G). To confirm that the loss of ciliary band neurons in Onecut
morphants is not because of a developmental delay resulting from
the morpholino injection, we show that these neurons remain ab-
sent from the ectoderm of Onecut morphants even at 4 d post-
fertilization (early larval stage) (Fig. 2H). We additionally show
that perturbing the function of Onecut does not broadly respecify
the ectoderm, as another cell type that is marked by the expres-
sion of gcm is expressed normally in these morphants (Fig. S6).
We also demonstrate that the sea star ortholog of Soxc is required
for elav expression. In Soxc morphants at 3 d following fertil-
ization, a loss of elav-positive cells was observed from both the
anterior pole territory and ectoderm near the developing ciliary
bands (Fig. 2I).
Taken together, these data suggest that a suite of genes, which
we term the pan-neurogenic suite, are first expressed within the
sea star fertilized egg, are restricted by cWnt signaling, and initiate
a regulatory program for the specification of a broad neurogenic
potential ectodermal territory. We do not know yet if these factors
are directly involved in the neural differentiation. However, the
pan-neurogenic suite, which includes at least onecut-, soxb1-, and
soxc-expressing cells, forms throughout the ectoderm and are
therefore not regulated by AP or DV patterning mechanisms
within the ectoderm. In this respect, the sea star develops much
like Saccoglossus, which maintains a pan-neurogenic ectoderm
throughout gastrulation (20). It has been argued that the ectoderm
of sea urchins will become broadly neurogenic in the absence of
signaling (33); but, in normal development, this neurogenic po-
tential is rapidly restricted to the anterior pole territory (34, 35)
and ciliary band domain (16, 17). The presence of a broad neu-
rogenic potential ectoderm, therefore, may be a basal feature of
all echinoderms and hemichordates maintained in common for
almost 800 million y (36), although the particular details of the
GRN associated with this may differ. The striking difference is
that during development, elav-expressing differentiated neurons
become localized to ciliary bands and the anterior ganglia in
echinoderms, but in Saccoglossus differentiated neurons remain
throughout the ectoderm.
Patterning a Foxg-Defined Ciliary Band Domain. To understand how
the broad neurogenic potential ectoderm nonetheless leads to the
localized expression of elav, we next considered the role of AP and
DV patterning mechanisms in sea stars. We have shown previously
initially localized within one ectodermal domain on the ventral side of
43-h-old gastrulae. (B) By 69 h, foxg transcripts are cleared within the sto-
modeal (mouth) ectoderm and remain within in a single domain that forms
around the mouth and loops over the anterior pole. (C) In 83-h-old larvae,
transcripts of foxg are clearly localized within the ectoderm of both ciliated
bands. (D) Transcripts of onecut localize to the ectoderm of the ciliary bands
in ∼72-h-old larvae. (E) Around 72 h, transcripts of onecut are found
throughout the ectoderm of Foxg morphants (MO). (F) Expression of foxg is
lost from the ectoderm in Nodal morphants around 48 h. (G) Anterior pole
view (APV) shows foxg transcripts within only the ventral ectoderm. (H and I)
In Bmp2/4 morphants, foxg transcripts are detected within a concentric
domain around the middle of gastrulae. (I) APV of foxg expression in Bmp2/4
morphants. (J) In Bmp2/4 morphants at around 96-h postfertilization, foxg is
expressed in two concentric loops above and below the mouth that corre-
spond to the pre- and postoral ciliary bands, respectively; foxg expression
does not extend into the anterior pole ectoderm. (K) Transcripts of six3 lo-
calize within the anterior ectoderm at 48 h. (L) Transcripts of wnt8 localize
within the posterior ectoderm at 48 h, and (M) expand anteriorly in Six3
morphants. (N) Ventral view of foxg expression within the ectoderm of
the pre- and postoral ciliary bands, above and below the mouth (Mo), re-
spectively. (O) Ventral view of foxg expression shows that preoral ciliary
band above the mouth shifts anteriorly in Six3 morphants. Vertical brack-
eted bars in (N and O) highlight the reduction in the anterior oral hood
ectoderm of Six3 morphants compared with controls. (P) Transcripts of nk2.1
within the ventral anterior pole ectoderm at the gastrula-stage. (Q) Ex-
pression of nk2.1 expands dorsally in Bmp2/4 morphants. (R) The tran-
scriptional effector of BMP2/4 signaling, phosphorylated Smad1/5/8 (pSmad1/5/8)
is localized throughout the dorsal ectoderm (pink); DAPI staining shows
nuclei (blue). (Magnification: 200×.)
Patterning of a foxg-expressing CBD. (A) Transcripts of foxg are
Yankura et al.PNAS
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that there are at least four molecularly distinct AP restricted
domains within the sea star ectoderm that are similar to orthol-
ogous domains observed within the hemichordate ectoderm and
the developing vertebrate nervous system (25). For example, we
find concentric domains of expression of zic, foxq2/retinal homeobox
(rx), six3, and nk1 along the AP axis (25). Although these domains
are less apparent in the sea urchin, we have suggested previously
that at least some of these AP domains may exist and that they are
likely compressed into the anterior pole ectoderm much earlier in
In addition to AP domains, sea star gastrulae also have distinct
domains of gene expression within the ectoderm along the DV
axis. For example, we have previously shown that foxg and foxa
are expressed on the ventral surface of gastrulae (13, 25). We
also cloned nodal and bmp2/4 orthologs from P. miniata and ex-
amined their expression. Similarly to sea urchin, and as expected,
the expression of nodal (Fig. S7A) and bmp2/4 (Fig. S7B) is re-
stricted along the DV axis early in development, although by late
gastrulation bmp2/4 expression is no longer detected in the ecto-
derm. We additionally examined the localization of activated
Smad1/5/8, the transcriptional effector for BMP2/4 signaling.
Just as has been shown in sea urchins (15), we find that phos-
phorylated Smad1/5/8 is found throughout the dorsal ectoderm
of blastula (see, for example, Fig. 4). Again, by around 60 h of
development (late gastrula) we no longer detect pSmad1/5/8 by
immunohistochemistry. Thus, it is immediately evident that neu-
rogenesis can occur broadly throughout the ectoderm without
regard to the presence of active BMP signaling.
Of the DV restricted genes, the ectodermal expression of foxg
is quite different from that of other sea star regulatory genes that
are later expressed within the ectoderm of the ciliary bands. It is
first observed relatively much later in development and only on
the ventral side of early gastrulae (Fig. 3A) (25). As development
proceeds, foxg expression clears from the stomodeal (mouth) ec-
toderm and remains expressed within a mostly concentric domain
that appears not unlike the expression of its ortholog in the sea
urchin (37) (Fig. S8). A critical difference, however, is that foxg
expression in sea stars is detected shortly thereafter in a domain,
which we term the ciliary band domain (CBD), that starts to ex-
tend dorsally and anteriorly over the anterior pole (Fig. 3B). By
larval stage, foxg expression is restricted to both ciliary bands (Fig.
3C) in what we think is the edge of its former domain. We observe
a loss of localized onecut expression within the ectoderm of the
ciliary bands of Foxg morphants (compare Fig. 3 D and E). This
finding suggests that one of the roles of Foxg is to organize gene
expression within the ectoderm of the pre- and postoral ciliary
bands. Therefore, it is possible that the observed role of Onecut
in neurogenesis (Fig. 2 G and H) may occur only after its ex-
pression is restricted to the ciliary bands. The spatial relationships
of foxg, onecut, and elav within the ectoderm are summarized
in Fig. S8.
We next sought to understand how this foxg-CBD is shaped.
Expression of foxg within the ectoderm is lost in Nodal morphants
(compare Fig. 3 A and F, and Fig. S7), as is foxa expression within
the stomodeal ectoderm (Fig. S7). Thus, Nodal signaling establishes
two nested ectodermal domains on the ventral side of sea star
gastrulae, the foxg-expressing domain that includes and surrounds
the more restricted foxa-expressing stomodeal domain.
In addition, foxg is no longer polarized to the ventral surface of
gastrulae when Bmp2/4 signaling is disrupted using a targeted
morpholino. Expression of foxg is observed in a ring around the
middle of Bmp2/4 morphants (compare Fig. 3 A and G to H and I,
respectively); however, the AP boundary of foxg expression above
and below the mouth does not appear to change (compare Fig. 3 A
and H). This finding suggests that AP positioning of the foxg-CBD
domain is established independently of DV patterning mechanisms.
In 4-d-old Bmp2/4 morphants, expression of foxg is similarly radi-
alized, although foxg expression has properly resolved into distinct
pre- and postoral ciliary bands (Fig. 3J).
We next examined how AP patterning mechanisms may affect
foxg expression. In vertebrates, Six3 gene products repress Wnt-
mediated posteriorization of the neuroectoderm (38, 39). The
sea urchin ortholog of Six3 also has the potential to repress Wnt
signaling, because overexpression of Six3 represses the expression
of several wnts, although their expression remains unchanged in
Six3 morphants (35). In 2-d-old sea star gastrulae, six3 is expressed
in the anterior ectoderm (Fig. 3K), in a domain that seemingly
does not overlap with wnt8 expression within the posterior ec-
toderm (Fig. 3L). To determine if Six3 promotes anterior ecto-
dermal identity by repression of wnt8, we blocked the translation
of six3 using a specific translation blocking morpholino. When the
function of Six3 is disrupted, the expression of wnt8 expands an-
teriorly, suggesting that Six3 antagonism of Wnt signaling estab-
lishes a boundary between the anterior and posterior ectoderm
(compare Fig. 3 L and M) and that this mechanism is conserved
between these echinoderms and vertebrates. Consistent with this
finding, in Six3 morphants at 4 d postfertilization (early larval
stage) there is a morphologically obvious reduction in the anterior
region of larvae (i.e., the oral hood ectoderm above the mouth is
reduced). In addition, the archenteron, a structure that derives
from vegetal (posterior)-most epithelium and forms the endoderm
and mesoderm, appears elongated. Importantly, expression of foxg
indicates that the position of the preoral ciliary band above the
mouth shifts anteriorly in Six3 morphants; the position postoral
ciliary band below the mouth, as expected, remains relatively
unchanged (compare Fig. 3 N and O). Thus, shifting the Wnt8-
Six3 boundary that respectively demarcates the posterior versus
anterior ectoderm, results in an anterior shift in foxg expression.
Although the finer details of how the foxg-CBD extends up
through the anterior pole ectoderm are not yet understood, we
phants at about 96 h, elav-expressing ciliary band associated neurons are absent
from the lateral surface; however, elav-expressing ciliary band neurons remain
on the ventral surface (arrows in A) above and below the mouth (Mo). Expres-
sion of elav remains in the mesoderm (not in plane of focus). (B) A surface ec-
todermal view of A shows the presence of anterior dorsal elav-expressing
neurons in Foxg morphants. (C) In Bmp2/4 morphants at ∼96 h, loops of elav
expression within the ectoderm appear similar to foxg (see Fig. 3J). (D) elav ex-
pression within the ectoderm near the pre- and postoral ciliary bands, above and
below the mouth. (E) Expression of elav within the preoral ciliary band above
mouth shifts anteriorly in Six3 morphants similar to foxg (see Fig. 3O). Expression
of elav remains unchanged in the mesoderm (not in plane of focus in D and E).
Vertical bracketed bars in (D and E) highlight the reduction in the anterior oral
hood ectoderm of Six3 morphants compared with controls. (F) Transcripts of eph
are found throughout the ectoderm of the CBD around 72 h. (G) Transcripts of
efn are found throughout the non-CBD ectoderm in normal embryos, but (H)
throughout the ectoderm in Foxg morphants. (I) Preoral (dotted lines) and
postoral (arrows) elav-expressing ciliary band neurons above and below the
mouth, respectively, around 72 h. The asterisk marks elav-expressing anterior
dorsal neurons. (J) In ∼72-h-old Eph morphants, elav-expressing ciliary band
neurons are largely absent from the lateral surface; however, elav-express-
ing ciliary band neurons remain on the ventral surface (arrows) and within
the anterior, dorsal ectoderm (dotted line). (Magnification: 200×.)
Linking the Foxg-CBD to localized neural patterning. (A) In Foxg mor-
| www.pnas.org/cgi/doi/10.1073/pnas.1220903110Yankura et al.
suggest that a change in Bmp2/4-mediated gene expression within
the anterior pole ectoderm might also be involved in the shaping of
the CBD in this territory. We find that nk2.1 normally is expressed
within the anterior pole ventrally to the domain of foxg expression
in this territory. In Bmp2/4 morphants, nk2.1 is now expressed
throughout the anterior pole (compare Fig. 3 P and Q) and foxg
expression concomitantly fails to move into the anterior (Fig. 3).
It is possible that DV restriction of the gene expression within the
anterior pole ectoderm permits the extension of foxg expression
that is observed in late gastrulae. Thus, the normal processes of
ventralization and dorsalization of the anterior pole ectoderm
may allow for the extension of the CBD into this territory.
In summary, we have shown that the sea star first produces a
broad neurogenic potential ectoderm that is not initially pat-
terned along the DV or AP ectoderm axis. This broad neurogenic
potential ectoderm is then overlaid with a highly patterned CDB.
The AP and DV patterning mechanisms that shape the expres-
sion of this CBD and rely on interactions between Nodal, BMP2/4,
Wnt and Six3, at least, to establish this foxg-expression territory.
Linking Patterning to the Final Steps of Neurogenesis. What remains
to be shown is how the final differentiation to elav-expressing
neurons is achieved. Crucially, we show that elav-expressing cil-
iary band neurons, but not anterior neurons that will likely form
the dorsal ganglia, are largely absent from the ectoderm of foxg
morphants (Fig. 4 A and B). We note that this aspect is unlike
Soxc morphants, in which both ciliary band and anterior dorsal
neurons are absent (Fig. 2I). We find that the localization of elav-
expressing ciliary band neurons within the ectoderm also shifts
when the shape of the foxg-CBD is altered. The expression of
elav is radialized in two distinct rings above and below the mouth
in these Bmp2/4 morphants (Fig. 4C) and the positioning of the
elav moves anteriorly in Six3 morphants (compare Fig. 4 D and
E). Importantly, the presence of elav-expressing cells does not
appear to change in response to these perturbations, rather only
the location of elav-positive cells changes within the ectoderm.
Therefore, elav-expressing cells can form in many locations
within the ectoderm as long as they are associated with foxg and
only after foxg becomes localized into ciliary bands. Additionally
these elav-expressing cells only appear after phosphorylated
Smad1/5/8 is no longer detected within the ectoderm. However,
elav-positive cells do not overlap with foxg expression and only
form along the outer edge of its domain of expression and not,
for instance, within the former foxg-CBD territory (Fig. 1C). An
additional mechanism is required to allow final neural differentia-
tion and this is likely controlled by the action of some transcription
factor or signaling molecule that forms a boundary demarcated
by foxg expression. We observed that the ephrin receptor (eph) is
expressed throughout the foxg-CBD of late gastrulae (Fig. 4F),
but the ephrin ligand (efn) is expressed exactly opposite to this,
throughout the non-CBD ectoderm (Fig. 4G). The expression
of these genes therefore forms a border at the edge of the foxg
domain. In Foxg morphants, we observed a nearly ubiquitous
expression of efn throughout the ectoderm (Fig. 4H). Therefore,
an additional function of Foxg is to segregate out two molecularly
distinct ectodermal domains, such that there is a boundary of
gene-expression profiles; this is represented by, at least, the ap-
posed expression of eph and efn at the edge of its territory. In
vertebrates, interactions of Eph and Efn can mediate directed
cell migrations (40). Therefore, we suspect that one of the roles
of the Eph/Efn boundary in the sea star is to guide cells to a
closer association with the ciliary bands. In sea stars, Eph mor-
phants have a reduction of differentiated neurons along the
lateral parts of the ciliary bands (compare Fig. 4 I and J), which
could suggest that cell migration is needed to bring prediffer-
entiated cells from the pan-neurogenic ectoderm to a closer
association with the foxg-CBD before they can express elav. It is
also possible that the Eph and Efn are simply involved in estab-
lishing two distinct territories that are needed to permit final
differentiation and have no direct role in neural migrations. At
present, we do not know how many times soxc-expressing neural
precursor cells divide and thus, for now, cannot compare numbers
of soxc versus elav-expressing cells to help distinguish between
these alternatives. This is a direction of further study.
This study was driven by the important need to understand the
developmental mechanisms that lead to a complex distribution
of neurons across the ectoderm of a dipleurula-like larva, which
is considered the basal larval type of deuterostomes. The data
presented here clearly explain how elav-expressing cells are
patterned across the AP and DV axes in association with the two
ciliary bands in sea stars (summarized in Fig. 5 and Fig. S9). The
sea star first likely uses maternally inherited anisotropies and the
cWnt pathway to distinguish endomesoderm from neurogenic ec-
toderm that persists through gastrulation. This endomesoderm is
then overlaid with a foxg-patterning module, which is shaped by
Nodal signaling from the mouth and is patterned by Bmp2/4 and
Six3. Foxg, in turn, is needed to form two distinct ciliary bands at
the edge of this domain and to distinguish a molecular boundary
between the CBD and other ectoderm. Thus, significantly, Bmp2/4
signaling in sea stars does not distinguish differentiated neurons
endomesoderm (EM, blue) from ectoderm (Ecto, tan). A suite of neurogenic genes including onecut-, otxßb-, and soxb1 are expressed throughout the ec-
toderm of (A) blastulae and (B) gastrulae. (B) Delta Notch signaling patterns distinct soxc-expressing cells within the broad neurogenic potential ectoderm.
Nodal signaling establishes foxg and foxa expression within the ventral ectoderm (dark purple). (C) During late gastrulation, foxg expression clears from the
stomodeal (mouth) ectoderm and a foxg-CBD (light purple) is shaped by Bmp2/4 signaling and Six3. The creation of this foxg-CBD, distinguishes between eph-
and efn-expressing ectodermal territories. Differentiated neurons (magenta) form along this boundary. Dotted lines in C denote the position of the foxg-CBD
within the ectoderm along the AP axis; six3-expressing anterior ectoderm extends upward from the bottom dotted line and includes the anterior pole
ectoderm, above the top dotted line.
Model for localized patterning of neurons within a broad neurogenic potential ectoderm. (A) Canonical Wnt (cWnt) signaling initially establishes
Yankura et al.PNAS
| May 21, 2013
| vol. 110
| no. 21
from nonneuronal ectoderm but instead contributes to the pat-
terning of the CBD, which then in turn provides cues to permit the
final steps of neuronal differentiation. The final fate commitment to
neuronal cell type in sea star may instead be governed by a temporal
loss of BMP signaling rather than a spatial gradient as observed in
many other taxa. This functional modulation between specification
and patterning likely reflects the evolutionary history of this
GRN, in which an ancestral module for specification of a broad
neurogenic potential ectoderm was subsequently overlaid with
a GRN for patterning. Such patterning could allow a swimming
larval form to more efficiently coordinate ciliary beating.
Embryo Culture and Characterization of P. miniata Gene Expression. Embryos
were cultured in seawater at 15 °C and fixed for in situ hybridization, as pre-
viously described (26). Partial sequences of P. miniata orthologs were obtained
via screening a P. miniata arrayed cDNA library (26) or were obtained from
the publically available transcriptome. Whole-mount in situ hybridization
(WMISH) and FISH was performed as previously described (25, 26).
qPCR and NanoString nCounter Assay. RNA from developmentally staged
P. miniata embryos was extracted using GenElute Mammalian Total RNA Kit
(Sigma-Aldrich). cDNA was synthesized using iScript Select cDNA Synthesis Kit
(Bio-Rad). Abundance of gene transcripts was determined using the nCounter
Gene Expression Assay (NanoString Technologies) as described by ref. 41 and
using custom P. miniata Reporter CodeSet and Capture ProbeSet.
Microinjection. Microinjection was performed as described previously (26).
Morpholino antisense oligonucleotides were designed by GeneTools. Ap-
proximately 20–40 injected embryos were used in spatial analyses of gene
expression. Experiments were repeated at least twice. Similar phenotypes
were observed when a second morpholino targeted to the same transcript
was used. The embryos presented are representative of changes observed in
greater than 90% of injected embryos.
ACKNOWLEDGMENTS. We thank Smadar Ben-Tabou de-Leon and Lynne
Angerer for critical reading of this manuscript and fruitful discussions;
James Fitzpatrick and Haibing Teng for technical advice; David McClay for
kindly providing the dominant-negative cadherin construct; two anonymous
reviewers for their thoughtful comments; Brenna McCauley for sharing data
about the temporal expression of several Patiria miniata orthologs; Adam
Foote for assistance with phylogenetic analysis; and Marinus Inc. and Pete
Halmay and Pat Leahy for animal collection. This work was partially supported
by National Science Foundation Grant 0844948 (to V.F.H.) and Howard Hughes
Medical Institute Undergraduate Education Grant 52006917 (C.S.K.).
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| www.pnas.org/cgi/doi/10.1073/pnas.1220903110Yankura et al.
Yankura et al. 10.1073/pnas.1220903110
Patiria miniata (Sea Star) Sequences. P. miniata cDNA sequences
were deposited in GenBank [Accession nos.: JX844799 (ephrin),
JX844800 (ephrin receptor), JX844801 (elav), JX844802 (soxb1),
JX844804 (soxc), JX844803 (wnt8), KC669537 (nodal), and
KC669538 (bmp2/4)]. Isolation of other P. miniata genes used
in this study are described elsewhere (1–4).
Morpholinos. The Delta morpholino sequence is published in
Other morpholino sequences used in this study were as follows:
PmOnecut 5′- TAGCTCGCTTGAAAGCATCACAAAC-3′;
1. Hinman VF, Nguyen AT, Davidson EH (2003) Expression and function of a starfish Otx
ortholog, AmOtx: A conserved role for Otx proteins in endoderm development that
predates divergence of the eleutherozoa. Mech Dev 120(10):1165–1176.
2. Yankura KA, Martik ML, Jennings CK, Hinman VF (2010) Uncoupling of complex regula-
tory patterning during evolution of larval development in echinoderms. BMC Biol 8:143.
3. Hinman VF, Davidson EH (2007) Evolutionary plasticity of developmental gene regulatory
network architecture. Proc Natl Acad Sci USA 104(49):19404–19409.
4. Otim O, Hinman VF, Davidson EH (2005) Expression of AmHNF6, a sea star orthologue
of a transcription factor with multiple distinct roles in sea urchin development. Gene
Expr Patterns 5(3):381–386.
of elav. Tree topology was determined using maximum likelihood and Bayesian analysis. Like the hemichordate, Saccoglossus kowalevskii, the sea star,
P. miniata, has only one transcribed elav gene that is likely to be the single ortholog of the four vertebrate elav genes. Bb, Branchiostoma belcheri (amphioxus);
Dm, Drosophila melanogaster; Dr, Danio rerio; Hs, Homo sapiens; Mm, Mus musculus (mouse); Pm, Patiria miniata (sea star); Sk, Saccoglossus kowalevskii
(hemichordate); Sp, Strongylocentrotus purpuratus (sea urchin).
Evolutionary relationships among elav orthologs. Phylogeny describes the evolutionary relationships of P. minatata (sea star) elav to other orthologs
Yankura et al. www.pnas.org/cgi/content/short/1220903110 1 of 5
Whole-mount in situ hybridization (WMISH). (A) Expression of elav within two rows above and below the mouth (Mo) of 83-h-old larvae. (B) Transcripts of elav
are detected in the lower lip (LL) and esophagus (ES), as well as in the mesodermally derived coeloms that are located on either side of the esophagus, which
are not in the plane of focus in A. It is not yet known if mesodermal-derived elav-expressing cells are neurons. In vertebrates, elav-like 1/huA is expressed in the
mesoderm, and elav-like 2/huB, elav-like 3/huC and elav-like 4/huD are expressed exclusively in neurons (1). It is likely that the different vertebrate orthologs
of elav have partitioned these roles in concert with gene duplications. (C) Arrows point to transcripts of elav within the anterior dorsal ganglia (DG) of a
96-h-old larva. (Magnification: 200×.)
Expression of PmElav. Sea star embryos are oriented with anterior pole up in ventral (VV) and dorsal (DV) views; h, hours postfertilization. (A–C)
1. Pascale A, Govoni S (2012) The complex world of post-transcriptional mechanisms: is their deregulation a common link for diseases? Focus on ELAV-like RNA-binding proteins. Cell Mol
Life Sci 69:501–517.
many of these genes continues following 24 h (see Fig. S4). Genes in green are abundant in fertilized eggs; transcripts of these genes are detected throughout
the ectoderm of late blastulae using WMISH (1, 2, 4). Genes that are later expressed within ectodermal domains along the AP axis (orange) of late blastulae (2)
are abundant between 12 and 16 h postfertilization (hpf) (i.e., early cleavage). Transcripts of nk2.1 (purple), which are later detected within a ventral ecto-
dermal domain of late blastulae (2), are abundant around 16–20 hpf. Transcripts of gcm (teal), which later have a spotted pattern of localization in late
blastulae (3), are abundant around 20 hpf. Also included in this schematic is the onset of expression of elav (magenta) within anterior neurons and ciliated
band (CB) neurons that surround the foxg-ciliated band domain (light purple). AP, anterior-posterior.
Temporal expression of sea star genes. Schematic summarizes the onset of gene expression during sea star development, although expression of
Yankura et al. www.pnas.org/cgi/content/short/12209031102 of 5
was determined using a time-course cDNA from fertilization (FE, fertilized egg) to 72 h postfertilization and qPCR. Cycle threshold (Ct) values at each time-
point are provided for each gene. Ct values are inversely related to transcript abundance. Based on our analyses of reverse-transcriptase minus samples, we
consider Ct values of less than 30 as not expressed or expressed at low enough levels to not be biologically relevant. Alternatively, or in addition to qPCR,
Nanostring nCounter assays provided transcript prevalence at the same developmental time points. NanoString counts (in italics) are provided for otxβb, bone
morphogenetic protein 2/4 (bmp2/4), six3, zic, foxq2, nk2.1, and wnt8. Here, values less than 200 counts are considered as not expressed or expressed at low
enough levels to not be biologically relevant. UD, undetected. Colors indicate when corresponding genes are highly expressed.
Abundance of P. miniata orthologs as determined by quantitative RT-PCR (qPCR) and NanoString. Temporal expression of sea star, P. miniata, orthologs
of onecut (A), otxßb (B), and elav (C) are found throughout the ectoderm of embryos in which cWnt signaling is blocked via injection of Δ-cadherin RNA. All
panels are WMISH. (Magnification: 200×.)
Canonical Wnt (cWnt) signaling establishes a neuroectoderm from endomesoderm. Embryos are oriented with the anterior pole up. Transcripts
Both panels are WMISH. (A) Transcripts of glial cells missing (gcm) are detected within distinct ectodermal cells throughout the ectoderm in ∼48-h-old gastrulae.
(B) Expression of gcm remains unchanged in Onecut morphants (MO).
Expression of glial cells missing (gcm) within the ectoderm does not change in Onecut morphants. Embryos are oriented with the anterior pole up.
Yankura et al. www.pnas.org/cgi/content/short/1220903110 3 of 5
left, except when indicated otherwise. All panels are WMISH. Transcripts of nodal (A) and bmp2/4 (B) are localized to the ventral side of blastulae. Transcripts
of foxa are localized within the endoderm and in a single ventral ectodermal domain (dotted lines) in 48-h (C) and 72-h (D) gastrulae. Expression of foxa is
lost from the ectoderm of Nodal morphants (MO) at around 48 h (E) and 72 h (F, ventral view); foxa expression remains within the endoderm as in normal
development. In Nodal morphants (MO) at around 72 h (G) and 96 h (H, ventral view), expression of foxg is lost from the ectoderm; foxg expression remains
within a mesodermally derived coelom (not in plane of focus in G; arrow in H) as in normal development. (Magnification: 200×.)
Nodal signaling establishes foxa and foxg expression on the ventral side of gastrulae. Embryos are oriented with the anterior pole up. Ventral is to the
Ventral is to the left, except when indicated otherwise. Panels A–G are WMISH; panels H and I are FISH. (A) Ectodermal expression of foxg is initially localized
to a single ventral domain. (B) Expression of foxg clears from the stomodeal (S) ectoderm and begins to extend into the anterior pole domain. (C) By 69 h, the
expression of foxg is observed in a single CBD that forms around the mouth and loops over the anterior pole. (E) By 83 h, foxg is clearly expressed within
the ectoderm of the ciliary bands, at what we think is the edge of its former domain. (F) During the time when foxg expression is restricted to the ventral
ectoderm, the expression of onecut is pan-ectodermal. (G) Around 72 h, the expression of onecut is localized to the ectoderm of the ciliary bands, which form
at the edge of the foxg-CBD. (H) By 96 h, expression of onecut within the ciliary bands is similar to that of foxg (compare with D). elav-expressing cells (green,
see arrows) do not colocalize with foxg expression (pink) in the CBD (H) or in the ciliary bands (I, ventral view); anterior neurons (asterisk in H). (J) Schematic
summarizes the spatial relationships among domains of foxg, onecut, and elav expression during development. (Magnification: 200×.)
Spatial relationships among foxg-expressing ciliary band domain (CBD), onecut, and elav expression. Embryos are oriented with the anterior pole up.
Yankura et al. www.pnas.org/cgi/content/short/12209031104 of 5
Fig. S9. Download full-text
genes that includes onecut, otxßb, and soxb1 establishes a broad ectodermal territory in which soxc-expressing neural precursors are patterned by Delta
Notch signaling (specification module, teal). Inputs from Onecut and Soxc are required for the specification of elav-expressing ciliary band neurons (magenta)
that are patterned across the AP and DV axes. An ectodermal foxg-expressing CBD (purple) is patterned along the AP and DV axes by conserved patterning
mechanisms (patterning module, tan). The foxg-CBD, possibly along with Ephrin receptor (Eph)/Ephrin ligand (Efn)-mediated directed cell migration, organizes
the formation of ciliary band neurons along the edge of the CBD within the broad neurogenic ectoderm.
Schematic of gene regulatory network interactions linking broad neural specification and localized patterning. A suite of pan-ectodermal regulatory
Yankura et al. www.pnas.org/cgi/content/short/12209031105 of 5