Six3 demarcates the anterior-most developing brain region in bilaterian animals.
ABSTRACT The heads of annelids (earthworms, polychaetes, and others) and arthropods (insects, myriapods, spiders, and others) and the arthropod-related onychophorans (velvet worms) show similar brain architecture and for this reason have long been considered homologous. However, this view is challenged by the 'new phylogeny' placing arthropods and annelids into distinct superphyla, Ecdysozoa and Lophotrochozoa, together with many other phyla lacking elaborate heads or brains. To compare the organisation of annelid and arthropod heads and brains at the molecular level, we investigated head regionalisation genes in various groups. Regionalisation genes subdivide developing animals into molecular regions and can be used to align head regions between remote animal phyla.
We find that in the marine annelid Platynereis dumerilii, expression of the homeobox gene six3 defines the apical region of the larval body, peripherally overlapping the equatorial otx+ expression. The six3+ and otx+ regions thus define the developing head in anterior-to-posterior sequence. In another annelid, the earthworm Pristina, as well as in the onychophoran Euperipatoides, the centipede Strigamia and the insects Tribolium and Drosophila, a six3/optix+ region likewise demarcates the tip of the developing animal, followed by a more posterior otx/otd+ region. Identification of six3+ head neuroectoderm in Drosophila reveals that this region gives rise to median neurosecretory brain parts, as is also the case in annelids. In insects, onychophorans and Platynereis, the otx+ region instead harbours the eye anlagen, which thus occupy a more posterior position.
These observations indicate that the annelid, onychophoran and arthropod head develops from a conserved anterior-posterior sequence of six3+ and otx+ regions. The six3+ anterior pole of the arthropod head and brain accordingly lies in an anterior-median embryonic region and, in consequence, the optic lobes do not represent the tip of the neuraxis. These results support the hypothesis that the last common ancestor of annelids and arthropods already possessed neurosecretory centres in the most anterior region of the brain. In light of its broad evolutionary conservation in protostomes and, as previously shown, in deuterostomes, the six3-otx head patterning system may be universal to bilaterian animals.
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ABSTRACT: Urchin embryos continue to prove useful as a means of studying embryonic signaling and gene regulatory networks, which together control early development. Recent progress in understanding the molecular mechanisms underlying the patterning of ectoderm has renewed interest in urchin neurogenesis. We have employed an emerging model of neurogenesis that appears to be broadly shared by metazoans as a framework for this review. We use the model to provide context and summarize what is known about neurogenesis in urchin embryos. We review morphological features of the differentiation phase of neurogenesis and summarize current understanding of neural specification and regulation of proneural networks. Delta-Notch signaling is a common feature of metazoan neurogenesis that produces committed progenitors and it appears to be a critical phase of neurogenesis in urchin embryos. Descriptions of the differentiation phase of neurogenesis indicate a stereotypic sequence of neural differentiation and patterns of axonal growth. Features of neural differentiation are consistent with localized signals guiding growth cones with trophic, adhesive, and tropic cues. Urchins are a facile, post-genomic model with the potential of revealing many shared and derived features of deuterostome neurogenesis. © 2014 Wiley Periodicals, Inc.genesis 02/2014; · 2.04 Impact Factor
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ABSTRACT: In bilaterians, which comprise most of extant animals, microRNAs (miRNAs) regulate the majority of messenger RNAs (mRNAs) via base-pairing of a short sequence (the miRNA "seed") to the target, subsequently promoting translational inhibition and transcript instability. In plants, many miRNAs guide endonucleolytic cleavage of highly complementary targets. Because little is known about miRNA function in nonbilaterian animals, we investigated the repertoire and biological activity of miRNAs in the sea anemone Nematostella vectensis, a representative of Cnidaria, the sister phylum of Bilateria. Our work uncovers scores of novel miRNAs in Nematostella, increasing the total miRNA gene count to 87. Yet only a handful are conserved in corals and hydras, suggesting that microRNA gene turnover in Cnidaria greatly exceeds that of other metazoan groups. We further show that Nematostella miRNAs frequently direct the cleavage of their mRNA targets via nearly perfect complementarity. This mode of action resembles that of small interfering RNAs (siRNAs) and plant miRNAs. It appears to be common in Cnidaria, as several of the miRNA target sites are conserved among distantly related anemone species, and we also detected miRNA-directed cleavage in Hydra. Unlike in bilaterians, Nematostella miRNAs are commonly coexpressed with their target transcripts. In light of these findings, we propose that post-transcriptional regulation by miRNAs functions differently in Cnidaria and Bilateria. The similar, siRNA-like mode of action of miRNAs in Cnidaria and plants suggests that this may be an ancestral state.Genome Research 03/2014; · 13.85 Impact Factor
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ABSTRACT: During early brain development, the organisation of neural progenitors into a neuroepithelial sheet maintains tissue integrity during growth. Neuroepithelial cohesion and patterning is essential for orderly proliferation and neural fate specification. Neuroepithelia are regionalised by the expression of transcription factors and signalling molecules, resulting in the formation of distinct developmental, and ultimately functional, domains.Neural Development 07/2014; 9(1):18. · 3.37 Impact Factor
Six3 demarcates the anterior-most developing
brain region in bilaterian animals
Patrick RH Steinmetz1,6†, Rolf Urbach2†, Nico Posnien3,7, Joakim Eriksson4,8, Roman P Kostyuchenko5, Carlo Brena4,
Keren Guy1, Michael Akam4*, Gregor Bucher3*, Detlev Arendt1*
Background: The heads of annelids (earthworms, polychaetes, and others) and arthropods (insects, myriapods,
spiders, and others) and the arthropod-related onychophorans (velvet worms) show similar brain architecture and for
this reason have long been considered homologous. However, this view is challenged by the ‘new phylogeny’
placing arthropods and annelids into distinct superphyla, Ecdysozoa and Lophotrochozoa, together with many other
phyla lacking elaborate heads or brains. To compare the organisation of annelid and arthropod heads and brains at
the molecular level, we investigated head regionalisation genes in various groups. Regionalisation genes subdivide
developing animals into molecular regions and can be used to align head regions between remote animal phyla.
Results: We find that in the marine annelid Platynereis dumerilii, expression of the homeobox gene six3 defines the
apical region of the larval body, peripherally overlapping the equatorial otx+ expression. The six3+ and otx+
regions thus define the developing head in anterior-to-posterior sequence. In another annelid, the earthworm
Pristina, as well as in the onychophoran Euperipatoides, the centipede Strigamia and the insects Tribolium and
Drosophila, a six3/optix+ region likewise demarcates the tip of the developing animal, followed by a more posterior
otx/otd+ region. Identification of six3+ head neuroectoderm in Drosophila reveals that this region gives rise to
median neurosecretory brain parts, as is also the case in annelids. In insects, onychophorans and Platynereis, the otx
+ region instead harbours the eye anlagen, which thus occupy a more posterior position.
Conclusions: These observations indicate that the annelid, onychophoran and arthropod head develops from a
conserved anterior-posterior sequence of six3+ and otx+ regions. The six3+ anterior pole of the arthropod head
and brain accordingly lies in an anterior-median embryonic region and, in consequence, the optic lobes do not
represent the tip of the neuraxis. These results support the hypothesis that the last common ancestor of annelids
and arthropods already possessed neurosecretory centres in the most anterior region of the brain. In light of its
broad evolutionary conservation in protostomes and, as previously shown, in deuterostomes, the six3-otx head
patterning system may be universal to bilaterian animals.
The brains of annelids and arthropods are similarly
composed of cerebral ganglia located above the foregut
and a variable number of associated segmental ganglia,
incorporated to the brain through cephalisation [1,2]. In
annelids, the cerebral ganglia develop, at least in their
largest part, from the neuroectoderm of the prosto-
mium, the most anterior part of the annelid body. In
polychaete annelids with indirect development, the pros-
tomium forms from the larval episphere, the upper half
of the trochophora larva (the apical “cap” anterior to the
primary trochoblasts forming the prototroch ciliary ring)
(Figure 1b). A smaller subset of cerebral neurons forms
from the peristomium, the more posterior part of the
developing head that contains the mouth and lies ante-
rior to the first metameric segment. The peristomium
forms from the equatorial larval regions including the
* Correspondence: email@example.com; firstname.lastname@example.org-
† Contributed equally
1Developmental Biology Unit, European Molecular Biology Laboratory,
Meyerhofstrasse 1, 69012 Heidelberg, Germany
3Johann-Friedrich-Blumenbach-Institute of Zoology, Anthropology and
Developmental Biology, DFG Research Centre for Molecular Physiology of
the Brain (CMPB), Georg August University, von-Liebig-Weg-11, 37077
Full list of author information is available at the end of the article
Steinmetz et al. EvoDevo 2010, 1:14
© 2010 Steinmetz et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
larval foregut (stomodaeum), the prototroch and meta-
troch ciliary bands if present (Figure 1b) [3,4].
In arthropods, the cerebral ganglia are composed of
the protocerebrum and two segmental neuromeres, the
deuto- and tritocerebrum. The most anterior part, the
protocerebrum, can be further subdivided into a more
lateral region bearing, for example, the optic lobes
(archicerebrum) and a median region that includes, for
example, the pars intercerebralis (prosocerebrum). Most
authors think that the archicerebrum represents the tip
of the neuraxis [1,5-8] but this has been disputed [9-11].
So far, it is unclear how the arthropod and annelid
brain parts are related, if at all, and how they would
align along the anterior-posterior axis [7,8,12,13]. In
order to molecularly reassess this long-standing ques-
tion, we have compared the expression of the anterior
regionalisation genes six3 and otx during the develop-
ment of annelid, arthropod and onychophoran brains.
Results and discussion
To elucidate head regionalisation in annelids (Figure
1b), we screened candidate genes for broad regional
expression in the larval episphere and, at later develop-
mental stages, in the prostomium. Previous studies iden-
tified molecular markers for sub-regions of the
episphere and prostomium (for example, Pdu-rx, Pdu-
nk2.1, Pdu-pax6) , for the equatorial ciliary girdle
and mouth region giving rise to the non-metameric
peristomium (Pdu-otx) [15,16], and for the posteriorly
adjacent larval segment giving rise to the segmented
trunk neuroectoderm (gbx  and hox ; Figure 1b).
In order to identify a broad regionalisation marker for
the anterior-most prostomium, we tested six3, because
in vertebrates the spatially restricted expression of this
gene demarcates the most anterior neural plate region
 and is required for the formation of anterior struc-
tures . six3 also demarcates the anterior body sec-
tion of the enteropneust Saccoglossus  (Figure 1c)
and of the sea urchin Strongylocentrotus purpuratus
larvae , consistent with a conserved role in the spe-
cification of the front end of the body. In the marine
annelid Platynereis dumerilii (Polychaeta, Phyllodocida),
Pdu-six3 (Additional file 1: Supplementary Figure 1a)
indeed proved to be a specific marker for almost the
entire episphere, expressed at early (Figure 2a, c, d) and
late larval stages (Figure 2e and Additional file 1: Sup-
plementary Figure 2a, c). None of more than 100 other
transcription factors tested showed a similarly broad
and contiguous episphere-specific expression ( and
data not shown). The broad apical domain of Pdu-six3
expression (Figure 2a, c, d) includes the anlagen of the
antennae and palpae and is surrounded by the ring-like
peristomial expression of Pdu-otx  (Figure 2b-d,
Additional file 1: Supplementary Figure 2b, l), which
covers equatorial/peristomial larval regions and overlaps
with six3 in the periphery of the episphere (Figure 2d-f).
The developing prostomium thus includes six3+ and
six3+/otx+ co-expressing parts, while the peristomium
expresses otx only (Figure 1b). Both six3+ and otx+ cells
include neural progenitors and differentiating neurons
as evidenced by co-expression with differentiation mar-
kers at 48 hpf (data not shown). As the positions of the
mouth and eyes have often been used as landmarks to
align the annelid and arthropod body regions, we also
Figure 1 Conservation of anterior-posterior six3/optix-, gbx/unplugged and Hox-expressing territories in Bilateria. A conserved anterior-
posterior alignment of six3/optix-, otx/otd-, gbx/unplugged- and hox-expressing neuroectodermal regions in the hypothetical ancestral arthropod
(a), the annelid Platynereis (b), and the hemichordate Saccoglossus (c). (a) Arrow depicts the antero-posterior neuraxis pointing at the anterior-
most six3/optix-region as identified by the data presented here. Light grey in (b): developing parapodial appendages, in (c): gut. Dark grey:
mouth opening. Yellow: neuroectoderm not expressing any of the mentioned genes. Purple in (a, b): six3+/otx+ regions. All animals are oriented
with anterior to the top. (a, b): ventral views. (c): lateral view. ea: eye anlage. Arthropod schematic after [29,36-38,48-53], Platynereis and
Saccoglossus schematics after references in the text.
Steinmetz et al. EvoDevo 2010, 1:14
Page 2 of 9
tried to affiliate the origin of these structures to the six3
+ or otx+ regions. In Platynereis, Pdu-six3 is expressed
in the stomodaeal roof (Additional file 1: Supplemental
Figure 2 a, c), while the stomodaeal Pdu-otx expression
starts broadly and becomes more restricted to single
cells during later stages (Additional file 1: Supplemen-
tary Figure 2b, d). Thus, the stomodaeum is of mixed
quality, but has its opening clearly surrounded by the
otx+ peristomial region (Additional file 1: Supplemen-
tary Figure 2a, b, yellow arrowheads). At 24 hpf, the
meric larval eyes express Pdu-otx (Additional file 1: Sup-
plementary Figure 2l) but not Pdu-six3 (not shown).
While the early Pdu-six3+ region is almost devoid of
Pdu-otx expression, both genes overlap more broadly at
later larval stages (Figure 2a-d, Additional file 1: Supple-
mentary Figure 2c, d and data not shown) in brain
regions that include the Pdu-r-opsin+ adult eyes 
(Additional file 1: Supplementary Figure 2 l, m and data
not shown). Thus, otx expression is shared by all eyes in
Platynereis (as it is in Drosophila), while only a subset
expresses additional six3, for example the Platynereis
adult eyes (similar to the Drosophila compound eyes
that express and require six3/optix ).
To obtain independent evidence that six3 plays a con-
served role in outlining the most anterior head region in
annelids, we cloned and investigated the expression of
otx and six3 orthologs (Additional file 1: Supplementary
Figure 1) in the oligochaete annelid Pristina longiseta
that asexually reproduces by fission into chains of indi-
viduals that each regenerate a full anterior-posterior axis
. During early fission, both genes are expressed in
stripes at the putative anterior part of the newly forming
head in the middle of a segment (Figure 2g, h). At this
stage, we were technically not able to resolve whether
Plo-six3 lies anterior of Plo-otx. However, in later stages,
using the developing antennae for spatial reference, we
indeed observed a single patch of Plo-six3 expressing
cells at the tip of a newly forming individual (Figure 2i),
in front of otx expressing cells  (Figure 2k).
We next tested whether a similar sequence of six3+
and otx+ regions also hallmarks the anterior end of the
arthropod body (Figure 3). In the fly Drosophila, we
found that optix/six3 indeed lies anterior of, and partly
overlaps with, orthodenticle/otx expression at stage 6
(late blastoderm) and stage 11 (elongated germ band)
(Figure 3a-c). However, since anterior-posterior pattern-
ing in Drosophila is known as being evolutionarily modi-
fied, we studied the beetle Tribolium castaneum where
an otx gene ortholog forms part of a more ancestral ante-
rior patterning system . The expression of Tc-six3
(Additional file 1: Supplementary Figure 1a) demarcates a
region at the tip of the germ rudiment , anteriorly
adjacent to the expression region of Tc-otd1 (Figure 3d),
Figure 2 Expression of annelid otx and six3 genes. In the
polychaete annelids Platynereis (a-f) and Pristina (g-k), six3
orthologues (a, c-e, g, i) are expressed anterior of otx orthologues
(b-d, f, h, k). Single (a, b, e-k) or two-colour (c, d) whole-mount in
situ hybridisations. Twenty-four hours (a-d) or 48 h (e, f) old
Platynereis larvae. Pristina early (g, h) and late (i, k) fission stage.
Asterisks in (a, b) point out stomodaeal expression (out of focus).
Dashed line: Prototroch ciliary band. (c,d) Blue: nuclear DAPI stain.
(i, k) Dotted line: Boundary of two forming worms dividing by
fission; continuous line: Plo-six3/Plo-otx expression boundary. Arrows:
Tentacles protruding dorsally from the anterior tip of the forming
Steinmetz et al. EvoDevo 2010, 1:14
Page 3 of 9
which is the only beetle otx paralog expressed at early
stages . At the elongated germband stage, the Tc-six3
(Figure 3e) and Drosophila six3 (Figure 3b, c) expression
regions are very similar and remain located at the ante-
rior-median edge of the germband, including the labrum
(Figure 3b, e), anterior brain neuroectoderm (Figure 3b,
e) and corresponding neuroblasts (Figure 3c)  and is
later also found in the developing stomodaeal roof (not
shown). This result suggests that the role of six3 as a
regional specification gene for the formation of the most
anterior head and brain region, as shown in Drosophila
and vertebrates, is conserved throughout Bilateria
[19,30]. To validate evolutionary conservation of the
anterior six3 region in other panarthropods, we isolated
the six3 and otx orthologues (Additional file 1: Supple-
mentary Figure 1) from the centipede Strigamia mari-
tima (Stm-six3, Stm-otx) and from the velvet worm
Euperipatoides kanangrensis (Eka-six3, Eka-otx) and for
both species found six3 expressed in an anterior-median
region at the tip of the germband and at later stages
Figure 3 Expression of insect, centipede and onychophoran six3 and otx genes. In the fly Drosophila (a-c), the beetle Tribolium (d, e),
the centipede Strigamia (f, g), the onychophoran Euperipatoides (h-i’), six3/optix orthologues (a-f, h) are expressed in an anterior-median location,
while otx/orthodenticle orthologues (a-e, g, i) are expressed more posterior-laterally. Single (f-i’) or two-colour (a, b, d, e) whole-mount in situ
hybridisations. (a, b) Drosophila stage 6 (a) and 11 (b). (c) Schematics of six3 (blue) and otx (red) neuroectodermal expression in the left head
hemisphere of a stage 11 Drosophila; expression of both genes is also detected in the underlying brain neuroblasts . (d, e) Tribolium germ
rudiment (d) and early elongating germband (e) stages. (f, g) Strigamia early segmentation stages. (h-i’) Euperipatoides mid-segmentation stages.
(h’, i’): nuclear SYBRGreen stain of embryos in (h, i) for better visualization of the mouth opening. Dotted line in (e): Anterior labral border. Blue
arrows in (b, e): six3+ neuroblasts. Dashed/dotted lines in (f, g): anterior germband margin. Yellow arrowheads in (h-i’): mouth opening.
Abbreviations: a = anterior, AN = antennal segment, CL = clypeolabrum, d = dorsal, DC = deutocerebrum, FG = foregut, Lr = labrum, MD =
mandibular segment, p = posterior, PC = protocerebrum, TC = tritocerebrum, v = ventral. Thin dashed line in (c): midline; thick dotted lines in
(c): posterior borders of the protocerebrum, deuterocerebrum and tritocerebrum. (a): Lateral view. (b-g): Ventral views. (h-i’): Ventro-lateral views.
All embryos with anterior to top except a: anterior to left.
Steinmetz et al. EvoDevo 2010, 1:14
Page 4 of 9
(Figure 3f, h and Additional file 1: Supplementary Figure
2e, g, i), while otx is mostly confined to more posterior
and lateral coordinates (Figure 3g,i and Additional file 1:
Supplementary Figure 2f, h, k). In Euperipatoides, the
Eka-six3 domain includes the antennal anlagen, while the
eye anlagen, as in other panarthropods, lie within the
more lateral Eka-otx+ domain (Figure 3h-i’, Additional
file 1: Supplementary Figure 2i, k) [31,32]. As in Platyner-
eis and Drosophila (Figure 3b), the mouth opening lies
within a ventral patch of otx expressing cells (Figure 3i, i’,
yellow arrowheads). At late Strigamia stages, the mouth
opening is broadly surrounded by six3 expression, but
also expresses otx at the posterior border (Additional file
1: Supplementary Figure 2g, h). For Euperipatoides and
Strigamia, the embryonic origin of the cells giving rise to
the mouth is unclear.
What is the fate of the six3+ region in the diverse
groups? In vertebrates, one prominent site of six3 activ-
ity is the developing hypothalamus [18,33]. Since in Pla-
tynereis, Pdu-six3 expression broadly covers the medial
brain anlagen, it includes a large part of the early differ-
entiating neurosecretory cells recently identified in
the 48 hpf Platynereis brain anlage  (Additional
file 1: Supplementary Figure 2c and data not shown).
In insects, the neurosecretory pars intercerebralis and
pars lateralis also originate from an anterior-median
head position suggesting their origin from a six3-expres-
sing region [34,35]. To validate this, we mapped six3/
optix expression in the Drosophila head ectoderm and
in brain neuroblasts (Figure 3b, c and Figure 4) .
We indeed found that the Six3+ dorsal brain region
includes the developing Dchx1+ pars intercerebralis
(Figure 4a-a’’, d) and the Fas2+ pars lateralis (Figure 4b-
b’’, d), both also positive for the invaginating placode
marker Crumbs (Figure 4c, c’, d) . Thus, the anlagen
for the neurosecretory pars intercerebralis and pars
lateralis lie within the six3+ region (Figure 3).
Our comparative expression data shows that the develop-
ing annelid, arthropod and onychophoran heads com-
prise an anterior-most six3+ region and a more posterior
otx+ region. Both regions are overlapping to a variable
degree but show a clear anterior-to-posterior sequence,
allowing cross-phylum alignment of head regions. In
arthropods, the six3+ and otx+ head regions give rise to
the protocerebrum and to the eyes (Figure 1a). In anne-
lids, the six3+and otx+ regions cover the developing
prostomium and the peristomium, from which the cere-
bral ganglia and eyes (and chemosensory appendages)
develop (Figure 1b), but the six3/otx-based molecular
subdivision does not fully match the morphological parti-
tion. While neuroectodermal six3 is restricted to the lar-
val episphere and thus to the prostomium, the more
posterior/equatorial otx expression covers the peristo-
mium but also part of the prostomium where it overlaps
with six3. Our data thus align annelid cerebral ganglia
with arthropod protocerebrum (that is, the most anterior
part of the arthropod cerebral ganglia, see “Background”).
Many authors have argued that the most anterior
structures in the arthropod brain are the anterior-lateral
regions mainly consisting of the optic lobe [1,5-8].
These ocular regions largely coincide with the otx+
region (Figure 1a). Yet, the clear anterior location of the
six3+ region in the early embryos of diverse arthropods,
together with the role of six3 in defining the most ante-
rior structures in other phyla, strongly suggest that it is
this median six3+ region, giving rise to the neurosecre-
tory pars intercerebralis and pars lateralis that repre-
sents the most anterior extreme of the arthropod brain
(arrow in Figure 1a) and corresponds to the neurosecre-
tory brain parts in annelids. This has hitherto been a
minority view [9-11]. As the terms “archicerebrum” and
“prosocerebrum” are tightly connected with the Articu-
lata theory - unsupported by almost all molecular phylo-
genies - and have been inconsistently used to include
different brain regions, we suggest abandoning these
terms. Instead, our comparative studies reveal the exis-
tence of a conserved, ancient neurosecretory brain part
at the tip of the neuraxis (Figure 1). It is followed by a
more posterior part of the head (and brain) anlage
expressing otx that often exhibits an early ring or arc-
like pattern [29,37,38], consistent with the radial head
hypothesis , and includes the eye anlagen (Figure 1).
In the animals investigated, the position of the mouth
opening is not reliably connected to the six3 or otx
region: while it comes to lie within the otx region of
Platynereis and onychophorans, its origin in arthropods
is unclear. The fact that the annelid and onychophoran
antennae develop from the six3+ region, in contrast to
the arthropod antennae that develop posterior to the otx
+ protocerebrum, is consistent with the previous
assumption of homology between annelid and onycho-
phoran antennae, but not with arthropod antennae .
The striking overall evolutionary conservation of a six3+
region in front of otx+ and hox+ regions in protostomes
documented here (Figure 1), as well as in vertebrates
and hemichordates, indicates that this anterior-posterior
series may be universal to bilaterian animals.
Animal culture and collecting
Platynereis larvae obtained from an established breeding
culture at EMBL, Heidelberg. Strigamia maritima eggs
collected at Brora, Scotland (June 2006). Fly strains: Ore-
gon R (wildtype). Female Euperipatoides kanangrensis
Reid, 1996 were collected from decomposing logs of
Eucalyptus trees in Kanangra Boyd National Park, NSW,
Steinmetz et al. EvoDevo 2010, 1:14
Page 5 of 9
Figure 4 The Drosophila six3/optix-expressing region includes neurosecretory centres. The neuroectodermal domains of the Drosophila
neurosecretory pars intercerebralis (PI) and pars lateralis (PL) lie within the six3/optix-expressing region. (a, a’, a’’) Six3/Dchx1 protein expression.
Six3 is detected in the neuroectoderm of the developing PI, as is specifically indicated by the expression of Dchx1. (b, b’, b’’) Six3/Fas2 protein
expression. Six3 is additionally found to be expressed in the neuroectodermal placode of the developing PL, as is indicated by the strong
expression of Fas2 . (c, c’) six3 mRNA/Crumbs protein expression. (c’) Higher magnification of the six3-expressing head region. Black
arrowheads in (c’) depict invaginating placodal cells of the PI (1) and PL (2) as visualized by apically concentrated localisation of the Crumbs
protein ; as is indicated by the red dots in (d). (d) Schematic summary of the expression of Six3, Dchx1, Fas2, and Crumbs in the anterior-
dorsal head ectoderm, including the neuroectodermal placodes of the PI and PL, as is depicted by the colour code. LR = labrum;
NE = neuroectoderm; OL = optic lobe anlagen; PI = pars intercerebralis; PL = pars lateralis.
Steinmetz et al. EvoDevo 2010, 1:14
Page 6 of 9
Australia (33° 59’S 150° 08’E). Females were kept in con-
tainers with dampened sphagnum moss at 13°C and were
fed crickets once every second week. Gravid females were
relaxed and killed with ethyl acetate vapour from Octo-
ber to December in order to acquire embryos of correct
stages. Embryos were dissected from the females in phos-
phate buffered saline (PBS) and, after removal of the egg
membranes, fixed in 4% formaldehyde in PBS overnight
at 4°C. Fixed embryos were dehydrated in a graded series
of methanol (25, 50, 75% in PBS with 0.1% Tween-20 for
10 minutes each) and stored in 100% methanol at -20°C.
Cloning of six3, otx and tryptophane-2,3-dioxygenase
All primers, PCR programs and template DNA source
are given in Additional file 2. Tc-six3 gene was identified
by in silico analysis of the Tribolium genome and ampli-
fied from a mixed stages (0 to 24h) cDNA library. Full
length Pdu-six3 was isolated by screening a 48 h l-ZAP
phage library (provided by C. Heimann, Mainz). Pdu-
tryptophane-2,3-dioxygenase gene was identified during
a sequencing screen of a 48 h Platynereis EST library.
Gene orthology was confirmed by using NCBI Protein
BLAST, MUSCLE  multiple sequence alignments
and CLUSTALX v.2 neighbour-joining phylogenetic
analysis  for complete proteins.
Database accession numbers
Eka-otx: EU347401, Eka-six3: EU347400, Plo-otx:
EU330201; Plo-six3: EU330202; Tc-six3: AM922337;
Stm-Six3: EU340980; Stm-otx: EU340979; Pdu-six3:
FM210809; Pdu-tryptophane-2,3-dioxygenase: FN868644
Whole-mount in situ hybridisation and
Established protocols were used for single- and two-colour
fluorescent whole-mount in situ hybridisations of Platy-
nereis and Pristina , Euperipatoides , Strigamia
, Drosophila , and Tribolium . A Drosophila
six3/optix RNA probe was synthesized from EST clone
LD05472 (Berkeley Drosophila Genome Project). Sub-
sequent immunostainings were done using Vector Red
(Vector Laboratories, Burlingame, CA, USA) or NBT/
BCIP (Roche Diagnostics Penzberg, Germany)). Primary
antibodies were: mouse anti-Crumbs (1:50; Developmental
Studies Hybridoma Bank, DSHB), mouse anti-Fas2 (1:20;
DSHB), rat anti-Orthodenticle  (1:1000, provided by
T. Cook), guinea pig anti-Dchx1 antibody (1:1000;
provided by T. Erclik), rabbit anti-Six3/Optix antibody
(1:300; provided by F. Pignoni), alkaline phosphatase
(AP)-coupled sheep anti-digoxygenin (1:1000, Roche). Sec-
AP-coupled donkey anti-mouse, Cy5-coupled goat anti-
rabbit (Dianova, Hamburg, Germany), Cy3-coupled goat
anti-mouse (Dianova, , Hamburg, Germany). SYBRGreen
(Invitrogen, San Diego, CA, USA) diluted 1:10.000.
Additional file 1: Supplementary figures and figure legends.
Steinmetz_Suppl_Figs.pdf contains two supplementary figures and
legends showing multiple sequence alignments of six3 and otx genes,
and supporting whole mount in situ hybridisation data of Platynereis,
Strigamia, and Euperipatoides larva.
Additional file 2: Supplementary methods. Steinmetz_SupplMethods.
xls is an Excel Spreadsheet containing primer sequences, template source
and PCR programs used to clone six3 and otx genes presented in the
AP: alkaline phosphatase; BCIP: 5-Bromo-4-Chloro-3’Indolyphosphate p-
Toluidine; DSHB: Developmental Studies Hybridoma Bank EST: expressed
sequence tags; otd: orthodenticle; NBT: Nitro-Blue Tetrazolium chloride; PBS:
phosphate buffered saline; PCR: polymerase chain reaction; PI: pars
intercerebralis; PL: Pars lateralis.
We thank Tiffany Cook (Cincinnati Children’s Hospital Medical Center) for
providing a Drosophila Orthodenticle-antibody. This work was funded by a
fellowship from the Luxembourg Ministry of Culture, Higher Education and
Research to P.R.H.S., by grants of the Deutsche Forschungsgemeinschaft
(DFG) to U.R. (UR 163/1-3, 1-4), by a grant of the Russian Foundation for
Basic Research (RFBR) to RPK (09-04-00866-a), through the DFG-Research
Center for Molecular Physiology of the Brain and BU-1443/2-2 to G.B, by a
BBSRC grant (BBS/B/07519) to C.B and by the Marie Curie RTN ZOONET
(MRTN-CT-2004-005624) to M.A. and D.A.
1Developmental Biology Unit, European Molecular Biology Laboratory,
Meyerhofstrasse 1, 69012 Heidelberg, Germany.2Johannes Gutenberg-
Universität Mainz, Institut für Genetik, J.-J.-Becher-Weg 32, 55128 Mainz,
Germany.3Johann-Friedrich-Blumenbach-Institute of Zoology, Anthropology
and Developmental Biology, DFG Research Centre for Molecular Physiology
of the Brain (CMPB), Georg August University, von-Liebig-Weg-11, 37077
Göttingen, Germany.4University Museum of Zoology, Department of
Zoology, Downing Street, Cambridge CB2 3EJ, UK.5Department of
Embryology, State University of St. Petersburg, Universitetskaya nab. 7/9,
199034 St. Petersburg, Russia.6University of Vienna, Department for
Molecular Evolution and Development, Althanstrasse 14, A-1090 Vienna,
Austria.7Vetmeduni Vienna, Institute of Population Genetics, Veterinärplatz 1,
A-1210 Vienna, Austria.8Queen Mary University of London, School of
Biological and Chemical Sciences, Mile End Road, London E1 4NS, UK.
PS analysed Platynereis six3 and otx expression, did multiple sequence
alignments, conceived further experiments and wrote the paper. RU
performed all Drosophila experiments. JE cloned and analysed Euperipatoides
six3 and otx genes. NP performed Tribolium gene expression experiments. RK
cloned and analysed six3 and otx genes in Pristina. CB cloned and analysed
Strigamia six3 and otx genes. KG analysed co-expression of Platynereis
tryptophane-2,3-dioxygenase and otx genes. MA and GB participated in the
design of the study and the writing of the paper. DA designed the study,
helped in writing the paper and cloned the Platynereis six3 gene.
The authors declare that they have no competing interests.
Received: 24 March 2010 Accepted: 29 December 2010
Published: 29 December 2010
Steinmetz et al. EvoDevo 2010, 1:14
Page 7 of 9
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Cite this article as: Steinmetz et al.: Six3 demarcates the anterior-most
developing brain region in bilaterian animals. EvoDevo 2010 1:14.
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