Current Biology 22, 2161–2166, November 20, 2012 ª2012 Elsevier Ltd All rights reserved
037
Deuterostomic Development
in the Protostome Priapulus caudatus
José M. Martı́n-Durán,1 Ralf Janssen,2 Sofia Wennberg,2
Graham E. Budd,2 and Andreas Hejnol1,*
1Sars International Centre for Marine Molecular Biology,
University of Bergen, Bergen 5008, Norway
2Department of Earth Sciences, Palaeobiology, Uppsala
University, Villavägen 16, Uppsala 75236, Sweden
Summary
The fate of the blastopore during development in the bilater-
ian ancestor is currently not well understood. In deutero-
stomes, the blastopore forms the anus, but its fate in
protostome groups is variable [1]. This variability, combined
with an absence of information from key taxa, hampers the
reconstruction of the ancestral developmental mode of the
Protostomia and the Bilateria. The blastopore fate of
the bilaterian ancestor plays a crucial role in understanding
the transition from radial to bilateral symmetric organisms
[2, 3]. Priapulids have a conservative morphology, an abun-
dant Cambrian fossil record, and a phylogenetic position
that make them a key group in understanding protostome
evolution [4, 5]. Here, we characterize gastrulation and the
embryonic expression of genes involved in bilaterian fore-
gut and hindgut patterning in Priapulus caudatus. We
show that the blastopore gives rise to the anus at the vegetal
pole and that the hindgut markers brachyury and caudal are
expressed in the blastopore and anus, whereas the foregut
markers foxA and goosecoid are expressed in the mouth in
the animal hemisphere. Thereby, gastrulation in the conser-
vatively evolving protostome P. caudatus follows strictly
a deuterostomic pattern. These results are more compatible
with a deuterostomic rather than protostomic (blastopore
forms the mouth) or amphistomic (mouth and anus are
formed simultaneously) mode of development in the last
common bilaterian ancestor.
Results and Discussion
The division of bilaterally symmetric animals (Bilateria) into the
Protostomia and the Deuterostomia [6] was traditionally based
on the fate of the blastopore [7], which is the orifice of invagi-
nation of the endomesodermal cells [8]. In deuterostomes, the
blastopore becomes the anus, whereas in protostomes the
fate of the blastopore is highly variable (Figure 1A and see
also Figure S1 available online). In some protostome lineages
the blastopore forms the mouth, in others it becomes the
anus, whereas in others it elongates and, by midclosure,
simultaneously forms the mouth and the anus (‘‘amphistomy’’)
[1, 2, 9]. The common shared fate of the blastopore in the Deu-
terostomia indicates that deuterostomy is the ancestral state
to this group. However, the diversity of blastopore fates
observed in the Protostomia makes it difficult to identify the
ancestral mode of development in this group. As a conse-
quence, not much is known regarding the fate of the
*Correspondence: andreas.hejnol@sars.uib.no
http://dx.doi.org/10.1016/j.cub.2012.09.
Report
blastopore in the last common bilaterian ancestor (Figure 1A).
The relationship between the blastopore, the mouth, and the
anus in the bilaterian ancestor plays a pivotal role in hypoth-
eses about the evolution of bilateral animals from an ancestor
with a single gut opening (as seen in extant cnidarians) [2, 3,
10, 11]. Therefore, reconstructing the ancestral fate of the blas-
topore in the Protostomia is a crucial step in understanding the
early evolution and diversification of the Bilateria.
Priapulid worms are a group of marine animals that belong
to the Ecdysozoa, which forms, together with the Spiralia
(Lophotrochozoa), the Protostomia. Most recent phylogenies
place priapulids in the third main group of ecdysozoans (the
Scalidophora; Figure S1), separate from the other two well-
studied lineages that include nematodes and arthropods,
respectively [6]. The adult priapulid is a sausage-shaped,
mud-dwelling, annulated worm of large size (0.5–20 cm), with
an anterior proboscis (or introvert) and a terminal mouth
(Figures 1B and 1C). Their fossil record indicates that the pria-
pulid body plan was among the most abundant and wide-
spread in the Early Cambrian [4, 12, 13]. Priapulids mostly
reproduce by external fertilization, and the embryos undergo
holoblastic radial cleavage [14], considered to be the ancestral
cleavage mode in ecdysozoans [15]. Correspondingly, nuclear
and mitochondrial genes tend to show high evolutionary
conservation in extant priapulids [5, 16]. In this way, priapulids
appear to be a conservatively evolving lineage and, as such,
a key group to consider for reconstructing the ancestral condi-
tion of the Ecdysozoa.
Gastrulation in the priapulid Priapulus caudatus starts at the
64-cell stage with the invagination of the vegetal-most cells
into a small blastocoel and the epibolic movement of the
animal blastomeres toward the vegetal pole [14]. Three days
after fertilization, the invaginated endomesodermal cells
define a small archenteron that opens to the exterior through
a blastopore in the vegetal pole (Figures 2A and 2A0 ), which
narrows by the end of gastrulation (Figures 2B and 2B0 ). Five
days after fertilization, the introvert-trunk boundary (Figures
1B and 1C) is established. Vegetal ectodermal cells below
the equator of the embryo invaginate and form an indentation
(Figure 2E) that divides the embryo into a larger animal and
a smaller vegetal hemisphere, which correspond to the intro-
vert and trunk of the larva, respectively (Figure 2C). Along
the upper ectodermal layer of the equatorial indentation, we
detect actin-positive cell outgrowths in seven cells (Figure 2E)
that will form the presumptive epidermal buccal tooth-like
structures of the larval introvert (‘‘scalids’’; Figure 1C). The
animal-vegetal axis of the egg thus corresponds to the ante-
rior-posterior axis of the larva and adult. We have named this
stage ‘‘introvertula’’. At this point, on the ventral (see below)
side of the introvert (animal hemisphere), a group of ecto-
dermal cells ingresses and forms the mouth of the embryo
(Figure 2C). The developing anus is identified by a strong
actin-positive bundle at the vegetal pole (Figure S2). After
6 days of development, the introvert-trunk boundary is more
pronounced (Figure 2D). Actin filaments in the introvert and
in the trunk indicate the developing musculature of the embryo
(Figures 2D and 2F). The mouth is now at an anterior terminal
position (Figure 2F), and the gut lumen runs from the mouth
Current Biology Vol 22 No 22
2162

A Bilateria
Protostomia Deuterostomia
Protostomy
blastopore mouth

Deuterostomy Deuterostomy
blastopore anus blastopore anus

Amphistomy
mouth
blastopore anus

Cnidaria Fly Roundworm Snail Earthworm Sea urchin Amphioxus Vertebrates

? D

B C tr
in

tr ca *

* in
sc

Figure 1. Fate of the Blastopore in the Bilateria and the Adult and Larva of Priapulus caudatus
(A) Diversity of blastoporal fates in the Bilateria. Whereas in the Deuterostomia the blastopore forms the anus, the Protostomia exhibits a diversity of
blastoporal fates. This diversity hampers the establishment of an ancestral fate of the blastopore to the Protostomia and, thus, to the last common bilaterian
ancestor. Nevertheless, deciphering the ancestral mode of development to the Bilateria is crucial to our understanding of the transition from animals with
a blind gut, like modern cnidarians, to bilateral symmetrical animals with a through gut.
(B and C) General appearance of an adult (B) (picture courtesy of www.kahikai.org) and a hatching larva (C) of the priapulid Priapulus caudatus. The annu-
lated body (cuticle rings pointed to by the white arrow in B) is divided into an anterior introvert and a posterior trunk separated by a ring-like constriction of
the cuticle (the introvert-trunk boundary, white arrowheads in B and C). The introvert can retract inside the trunk, which bears a caudal appendage of
unknown function at its posterior end in the adult. The mouth (asterisk) is located at the anterior end of the introvert and is surrounded by complex cuticu-
larized toothlike structures called scalids (C). The hatching priapulid gradually matures into the adult form through a series of molts. ca, caudal appendage;
in, introvert; tr, trunk; sc, scalids. Scale bars in (B) represent 1 cm and in (C) 50 mm.

to the anus, which is formed by four ectodermal cells at the
vegetal/posterior pole of the trunk (Figures 2D0 , 2F, and S2).
The direct formation of the anus from the blastopore
(deuterostomy) is supported by the embryonic expression of
the hindgut/posterior markers brachyury (bra) and caudal
(cdx) (Figure 3). Orthologs of bra are expressed in the blasto-
pore and gut opening of cnidarians; in the blastopore, foregut,
and hindgut of ambulacrarians, spiralians, and chaetognaths;
and in the blastopore and hindgut of arthropods (Table S1).
In P. caudatus, bra is expressed in the blastopore and in
a subset of endomesodermal cells during gastrulation (Figures
3A and 3B). In the introvertula stage, bra is only detected in
the anus (Figures 3C and 3D), which mirrors the lack of oral
brachyury expression in arthropods and suggests that this is
ancestral to the whole Ecdysozoa. The gene cdx is primarily
expressed in the hindgut of the deuterostomes and proto-
stomes (Table S1). The ortholog of cdx in P. caudatus is
widely expressed in the vegetal pole of the gastrula (Figures
3E and 3F), and in the trunk (posterior) of the introvertula
(Figures 3G and 3H).
The foregut markers foxA and goosecoid (gsc) are ex-
pressed during gastrulation in the animal hemisphere at the
site of the future mouth, before the ingression of ectodermal
cells becomes visible. Orthologs of foxA are expressed in the
pharynx of cnidarians and in the foregut, the endodermal
midgut, and the hindgut of deuterostomes and protostomes
(Table S1). During gastrulation, the priapulid ortholog of foxA
is expressed in a cluster of ectodermal cells on the ventral
side of the embryo (Figures 3I–3V) and in a subset of endome-
sodermal cells. At the introvertula stage, foxA is expressed in
the oral ectoderm and in the gut (Figures 3K and 3L). gsc is
expressed in the pharynx of cnidarians and in the foregut of
a broad range of deuterostomes and protostomes (Table
S1). In P. caudatus, gsc is detected in a ventral cluster of ecto-
dermal cells (Figures 3M–3Z) and in an anterior ectodermal
domain that includes the mouth of the introvertula (Figures
3O and 3P). Orthodenticle (otx) has a conserved function in
specifying anterior body regions and neural structures in
bilaterians (Table S1), and it is expressed in the presumptive
developing ventral nervous system of P. caudatus [17]
Deuterostomic Development in Priapulus caudatus
2163

Figure 2. Development, Gastrulation, and Early Organogenesis in Priapulus caudatus

z projections of confocal stacks of embryos at 3, 4, 5, and 6 days of development stained with phallacidin (green) and propidium iodide (magenta). Gastru-
lation (A and A0 ) occurs at the vegetal pole and involves the formation of a small archenteron and a conspicuous blastopore (asterisk). After the invagination
of the endomesoderm (B and B0 ), the archenteron collapses (white arrowhead in B), and the blastopore (asterisk) narrows. A high number of mitoses (white
arrows) in the ectoderm is observed, in particular in the animal hemisphere. At day 5 (C and C0 ), an inward ectodermal infolding appears at the vegetal pole
(white arrowheads in C and D), which marks the introvert-trunk boundary (magnified in E from the white rectangle in C). At 7 points of this infolding, an ecto-
dermal cell of the introvert region forms an actin-rich outgrowth (E) that will become an oral scalid. Note that the introvert shows a higher number of cells and
smaller cells than the trunk. The mouth is formed on the ventral side of the introvert independently from the site of gastrulation. (D, D0 , and F) After 6 days of
development, the mouth shifts to an anterior terminal position, and an actin-positive bundle that corresponds to the gut lumen (central phallacidin spot in F)
runs from the mouth to the anus. The former vegetal pole, where the blastopore opened, becomes the anus (D0 ). Schematic drawings of priapulid embryos at
days 3, 4, 5, and 6 of development (A00 –D00 ), respectively, placed in the general context of priapulid embryonic development. The black arrowheads indicate
the position of the introvert-trunk boundary and its movement during introvert retraction. Drawings are oriented with the animal/anterior pole to the top and
the ventral side to the left. Drawings are not to scale. In (A–E), animal/anterior to the top and vegetal/posterior to the bottom. In (F), the introvert projection is
to the top and the trunk to the bottom; in (C–D0 ), ventral is to the left. an, anus; ac, archenteron; bp, blastopore; dg, digestive tract; ec, ectoderm; em,
endomesoderm; mc, muscle; mo, mouth; sc, scalid. Scale bars in (A–D) and (F) represent 50 mm and in (A0 –D0 ) and (E) represent 10 mm.

(Figures 3Q–3T). The colocalization of foxA and gsc with
otx (Figures 3U–3AB) indicates that the mouth is formed
ventrally, a position that is considered to be ancestral in bilat-
erians [2, 3, 10, 11].
There is little consensus about the primary mode of gastrula-
tion of the Ecdysozoa, due to the diversity and divergence
observed in the main ecdysozoan lineages, namely the Nema-
toida (Nematoda + Nematomorpha) and the Panarthropoda
(Tardigrada + Onychophora + Arthropoda) [18, 19] (Figure S1).
Similar to P. caudatus, nematomorphs display holoblastic
cleavage and deuterostomy, with the blastopore forming in
the ventral-posterior region of the embryo [20]. Although nema-
todes show holoblastic cleavage, gastrulation and gut forma-
tion can occur either by amphistomy (e.g., Tobrilus stefanskii)
[19] or by the ventral-posterior ingression of the founder endo-
dermal cell(s) and subsequent closure of the blastopore (e.g.,
Enoplus brevis and C. elegans) [19, 21]. Therefore, the nemato-
morph and nematode conditions suggest that in the last
common ancestor of the Nematoida, gastrulation occurred in
the ventral-posterior region and the blastopore likely formed
posterior/anal structures (deuterostomy).
Tardigrades show holoblastic cleavage followed by gastru-
lation and blastopore closure [22, 23]. Onychophorans exhibit
mostly superficial cleavage [18], and gastrulation involves
either the closure of the blastopore [24] or amphistomy
[25, 26]. In accordance with their extreme ecological and
morphological diversity, divergent modes of cleavage and
gastrulation are observed among terrestrial hexapods, myria-
pods, and chelicerates [18]. On the other hand, the marine
pycnogonids and some crustaceans retain the putative ances-
tral holoblastic cleavage. Unlike in most arthropods and
ecdysozoans, the blastopore of pycnogonids forms in the
future dorsal side of the embryo and forms the mouth
(protostomy) or closes [15, 27]. Most crustaceans exhibit
deuterostomy with the blastopore forming in a ventral-poste-
rior position [18, 28, 29], which may be considered ancestral
to the Panarthropoda given the divergence of tardigrades,
onychophorans, and most arthropods and the similarity to
the development of the Nematoida and Priapulida.
Conclusions
We show deuterostomic development in P. caudatus,
a member of the third major lineage of the Ecdysozoa, the
Scalidophora. The expression of bra, cdx, foxA, gsc, and otx
during early development in P. caudatus is similar to that of
the nematodes and arthropods where it has been described
(Table S1). These findings indicate an ancestral ecdysozoan
expression pattern (Figure 4) and, together with the particular
Current Biology Vol 22 No 22
2164
conservatively evolving features of priapulids, strongly
suggest that the anus is the ancestral fate of the blastopore
in this major clade of protostomes.
Deuterostomy in the ecdysozoan ancestor has major evolu-
tionary implications. Given that chaetognaths [30] (an enig-
matic group of protostomes not belonging to the Ecdysozoa
nor the Spiralia [31, 32]) and deuterostomes also exhibit
deuterostomic development, it is likely that deuterostomy
was the ancestral developmental program in bilaterians (Fig-
ure 4). Furthermore, distinct and independent gene regulatory
networks to specify the blastopore, the anus, and the mouth
were present in the last common bilaterian ancestor, as it is
observed in extant priapulids, chaetognaths, acoels [33], and
deuterostomes (Table S1).
This scenario can easily explain the variability of fates of the
blastopore observed in the Protostomia by the spatial and/or
temporal coupling or decoupling of the blastoporal, oral, and
anal gene regulatory networks as new egg structures, modes
of cleavage, and reproductive strategies evolved. Accordingly,
protostomy is the secondary coupling of the genes specifying
the blastopore and the mouth, which appeared independently
in several lineages of ecdysozoans and the Spiralia. The
complete dissociation of the genes specifying the blastopore,
Figure 3. Expression of Hindgut and Foregut
Markers during Priapulus caudatus Gastrulation
and Mouth Formation
Whole-mount in situ hybridization in priapulid
embryos at the gastrula and introvertula stage.
During gastrulation, the hindgut markers bra
(A and B) and cdx (E and F) are expressed at the
vegetal pole and in the blastopore (magnified in
the insets of B and F). At the introvertula stage,
bra is expressed in the anus (C and D) and cdx
in the trunk posterior ectoderm (G and H). cdx
also shows expression on a small cluster of cells
of the introvert, probably associated with the
nervous system (black arrow in G). The foregut
markers foxA and gsc are expressed in an equa-
torial ectodermal domain of the gastrula (I and
J; M and N) (black arrows in I and M). At the intro-
vertula stage, foxA is expressed in the terminal
oral ectoderm of the introvert (inset in L) and in
the digestive tract (K and L), and gsc is expressed
in the mouth (inset in P) and in a lateral ecto-
dermal domain of the introvert (O and P). otx is
detected on one lateral of the priapulid gastrula
(black arrow in Q) in a U-shaped pattern (Q and R)
and in the anterior nervous structures of the in-
trovertula (S and T): one lateral of the introvert
(slashed line in S–AB), the mouth (inset in T),
and a ring around the introvert-trunk boundary
(black arrows in S, W, and AA). The simultaneous
detection of foxA and gsc with otx shows overlap-
ping expression patterns and, thus, indicates that
the specification of the mouth occurs in the
ventral side of the embryo. In all panels, the
asterisk indicates the vegetal/posterior region.
In (C), (G), (K), (O), (S), (W), and (AA), the black
arrowheads mark the introvert-trunk boundary,
and anterior is to the top and ventral to the left.
In (I), (M), (Q), (U), and (Y), ventral is to the left. In
(W), the white arrowhead points to the foxA
expression in the digestive tract. The schematic
drawings of the gastrula and introvertula stage
depict the reported expression domains. Draw-
ings are not to scale. The slight elongation in the
animal-vegetal axis of the embryos at the gastrula
stage is an artifact of fixation.
the mouth, and the anus during embryogenesis led to blasto-
pore closure, and, inversely, the simultaneous activation of
the mouth and anal gene regulatory networks in different
regions of the blastopore, together with the extension of the
embryo along the anterior-posterior axis, originated amphis-
tomy. This sequence of evolutionary events differs from tradi-
tional hypotheses about the origin of the Bilateria [2, 10, 11],
but agrees with the hypothesis that explains the transition
from radial to bilateral animals through the shift of the blasto-
poral gene regulatory network, from the animal to the vegetal
pole, that allowed the independent development of the meta-
zoan mouth from the blastopore in bilaterians [3]. The present
findings thereby challenge our classic ideas about the origin
and diversification of bilaterian animals and highlight the
importance of studying the great diversity of animal forms in
our pursuit of a better understanding of animal evolution.
Experimental Procedures
Adult gravid specimens of P. caudatus Lamarck 1816 were collected from
Gullmarsfjorden (Fiskebäckskil, Sweden) in November and spawned as
described previously [14]. Embryos were permeabilized with 0.05% thiogly-
colate and 0.01% pronase in filtered deep-sea water (FDSW) for 45 min at
Deuterostomic Development in Priapulus caudatus
2165

Bilateria Figure 4. Evolution of the Bilaterian Through Gut

* P. caudatus
Cnidaria Deuterostomia
(Nematostella vectensis)
Protostomia
* *
Ecdysozoa
(Ptychodera flava) (Priapulus caudatus)
- Separation of mouth development
from the blastopore (deuterostomy)
- Evolution of the through gut
Related to the Expression of bra, cdx, gsc, and
foxA and the Deuterostomic Development of
The expression of bra, cdx, gsc, and foxA indi-
* cates the homology between the mouth and the
anus of P. caudatus and other bilaterians, as
well as the homology of the oral opening of
cnidarians and the mouth of bilaterians. The
comparison of the embryogenesis and expres-
Spiralia sion data in P. caudatus with the other ecdyso-
(Platynereis dumerilii)
zoan lineages support considering deuterostomy
as the ancestral mode of development in the
- Evolution of protostomy Ecdysozoa and, therefore, suggest that the last
in certain groups
common ancestor to the Deuterostomia and Pro-
tostomia exhibited deuterostomic development.
This scenario differs from traditional hypotheses
that considered either protostomy or amphis-
bra tomy as the ancestral mode of development,
cdx but agrees with recent molecular results and
foxA a hypothesis that indicates the separation of the
gsc development of the mouth from the site of gastru-
lation as a key step in the evolution of the Bilate-
ria. Embryos are oriented with the animal pole
(also anterior, in bilaterians) to the top and the
ventral side (in bilaterians) to the left. The asterisk
indicates the fate of the blastopore in each
embryo. Only gene expression related to the
through gut is indicated in this diagram (see Table
S1 for references). Drawings are not to scale.

9 C and fixed in 4% paraformaldehyde in FDSW for 1 hr at room tempera-
ture. Actin filaments and nuclei were labeled with Bodipy-FL phallacidin
(Life Technologies) and propidium iodide (Sigma). Samples were cleared
in Murray’s reagent, and representative embryos were scanned in a Leica
SP5 confocal laser-scanning microscope. Genes were identified with
degenerate primers, and the full-length transcripts were amplified using
the Advantage2 RACE kit (Clontech). Gene orthologies were inferred using
Bayesian analyses (Figure S3). In situ hybridization was performed accord-
ing to Hejnol and Martindale [33]. Stained embryos were imaged with a Zeiss
Axiocam HRc connected to a Zeiss Axioscope A310 using bright-field
Nomarski optics. All images were analyzed with Fiji and Photoshop CS5
(Adobe).
Supplemental Information
Supplemental Information includes three figures, one table, and Supple-
mental Experimental Procedures and can be found with this article online
at http://dx.doi.org/10.1016/j.cub.2012.09.037.
Acknowledgments
The collection trips were funded by the European Union Infrastructures
program (ASSEMBLE grant agreement no. 227799). We thank K. Pang and
J. Ryan for improving the manuscript; B.C. Vellutini for the picture of a pria-
pulid larva; all the members of the S9 lab for help and discussion; and R.
Neves, F. Marlétaz, and all the people at the Sven Lovén Centre for Marine
Sciences for helping with the collections.
Received: July 29, 2012
Revised: September 2, 2012
Accepted: September 18, 2012
Published online: October 25, 2012
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