BACKGROUND
vertebrates possess pronounced visceral asymmetries along their left-right  body axis, although they belong to the large phylogenetic group of the bilateria, which refers to their bilaterally symmetric outer appearance  <cit> .
most organs are positioned in a characteristic way in the thoracic and abdominal cavities.
in all vertebrate species examined so far, these asymmetries are under the control of the nodal signaling cascade, which is only activated in the left lateral plate mesoderm before the first appearance of anatomical asymmetries  <cit> .
the secreted transforming growth factor beta  nodal binds to its receptor which results in the activation of its own transcription and that of its negative feedback inhibitor lefty , another secreted tgfβ superfamily member.
lefty antagonizes nodal signaling, providing its temporal and spatial control.
additionally, the paired-like homeodomain transcription factor pitx <dig> is induced downstream of nodal and mediates, through less well-known target gene activation, the setup of asymmetric organ morphogenesis.
the event activating this highly conserved developmental program is referred to as symmetry breakage.
even though variations of the common theme may exist, an ancestral mode of vertebrate symmetry breaking has emerged over the past few years: at the heart of this mechanism acts an extracellular leftward fluid flow, generated by a transient ciliated epithelium, the so-called left-right organizer  .
the vertebrate lro  in amphibians, and posterior notochord or ventral node in mammals) represents a field of mono-ciliated cells at the posterior end of the forming notochord, flanked by endodermal archenteron cells .
this unique tissue, which develops during early neurula stages, consists of superficially located mesendodermal cells which are transiently placed in the primitive gut or archenteron, where they function as the lro.
the cells of the lro subsequently leave the epithelium to ingress into deeper mesodermal structures  <cit> .
the polarized attachment of cilia at the posterior cell surface, together with their clockwise rotational movement, create an asymmetric stimulus by the leftward acceleration of extracellular fluids, i.e.
a leftward flow  <cit> .
this setup has been functionally described in mammals , amphibians  and teleost fish , and homologous tissues have been identified in salamanders  and basal bony fish .
interference with ciliary length, motility, polarization or flow function in general resulted in defects of the lr axis .
as a result of leftward flow, the nodal inhibitor dand <dig> becomes down-regulated on the left margin of the lro, where it is co-expressed on both sides with nodal itself.
nodal thus becomes liberated on the left side to induce the asymmetric signaling cascade in the left lateral plate mesoderm .
dand <dig> repression is induced through a flow-dependent intracellular calcium signal, which is mediated through the calcium channel pkd <dig> .
although ubiquitously expressed during these stages, pkd <dig> inhibition causes lr defects and prevents a unilateral calcium signal in mouse and fish lros .
outside of the vertebrate lineage, lr asymmetries are common as well.
for many deuterostome lineages, unilateral nodal expression has been described, which seems to be the common mediator of lr asymmetries in metazoan animals .
tunicates, the sister group of the vertebrates, as well as the cephalochordates, the most basal group of chordates, both express nodal asymmetrically on the left side  <cit> .
the latter seem to show an ancient state of this set-up, with many lro targets activated similarly to vertebrates, such as nodal, lefty, pitx <dig> and dand <dig> .
accordingly, interfering with nodal activity in the cephalochordate amphioxus also resulted in lr defects  <cit> .
the existence of an lro in amphioxus has been predicted, but not yet analyzed or functionally tested  <cit> .
within the deuterostomes, the ambulacraria, a chordate sister group which comprises the echinoderms, show unilateral nodal activation during late gastrula stages as well  <cit> .
although adult echinoderms took the path of reestablishing a radially pentameric body plan during evolution, their early embryo displays the typical bilaterally symmetrical embryonic development, which results in a pronounced lr asymmetry before metamorphosis  <cit> .
as in other deuterostomes, nodal is the first asymmetrically activated gene, and is first found on one side in the developing archenteron tip  <cit> .
the functional requirement and a complex downstream gene regulatory network have been elucidated in detail in sea urchin embryos  <cit> .
this asymmetric archenteron domain instructs a second symmetrical ectodermal nodal domain to switch towards this side as well  <cit> .
this expression is thought to be on the right, as deduced from a ventral mouth opening  <cit> .
we have previously argued that this difference can be resolved to a common evolutionary origin, if the ventral side of echinoderms is homologized with the dorsal side of chordates  <cit> .
this reasoning is based on the expression of dorsal organizer genes in the oral ectoderm of the sea urchin larva, suggesting that the mouth - counterintuitively - opens on the dorsal side.
such an evolutionary mouth repositioning can be easily envisaged as a transitional process from echinoderm ancestors to vertebrates  <cit> .
indeed, oral organizer identity has been recently shown to be functionally conserved in the sea urchin p. lividus  <cit> .
thus, in this scenario, left-asymmetric nodal expression is a synapomorphy of the deuterostomes  <cit> .
one major question, however, has remained unanswered: how does symmetry breakage upstream of asymmetric nodal induction occur in echinoderms?
do sea urchin embryos possess a lro or an evolutionary functional precursor that induces asymmetric nodal expression?
do archenteron cells possess cilia and, if so, are these required to induce asymmetric nodal expression?
using descriptive and functional approaches we show that  archenteron cells in the sea urchin larva harbor monocilia;  archenteron cilia are polarized and motile; and  cilia are required for asymmetric nodal induction.
RESULTS
sea urchin archenteron cells harbor polarized monocilia
the defining feature of vertebrate lros are polarized monocilia.
as an entry point into studying sea urchin symmetry breakage, we investigated the presence of cilia on staged gastrula embryos by performing immunofluorescence  with a well-characterized anti-acetylated α-tubulin antibody.
in addition to the previously described long ectodermal cilia, optical sections of gastrula stage paracentrotus lividus embryos revealed a population of shorter cilia within the developing archenteron .
higher magnification showed that most cilia ranged from 4–6 μm in length, with the most anterior ones being longer, measuring up to 10 μm .
the same basic characteristics of mesendodermal archenteron cilia were also found in a second sea urchin species, strongylocentrotus pallidus, demonstrating the conserved presence of cilia in the primitive gut of sea urchin larvae.
one slight variation was observed, namely that very early gastrula embryos of strongylocentrotus apparently lacked archenteron cilia , which were, however, present at mid to late gastrula stages .
in p. lividus, cilia were present already at earlier gastrula stages .fig.
<dig> polarized cilia at the sea urchin archenteron.
a–e
p. lividus  and s. pallidus embryos  were analyzed by if for the presence of cilia at the archenteron.
optical sections showed ectodermal  and archenteron cilia  at mid gastrula stages.
late  but not early s. pallidus gastrula stage embryos revealed cilia in the archenteron and at the archenteron tip .
cilia were stained with an antibody against acetylated-α-tubulin  or anti-α-tubulin , nuclei were stained with dapi , and cell boundaries were visualized by phalloidin-green  or phalloidin-red .
f, g sem analysis of p. lividus ectodermal and archenteron cilia.
fractured embryos allowed the visualization of monocilia on archenteron cells .
cilia are highlighted by an arrowhead  posterior polarization of cilia on cells which invaginated into the archenteron.
cilia are colored in yellow and individual cells alternating in green and purple.
the ciliary base is marked by a red semicircle, the center of the cell is indicated by a yellow dot.
schematic drawings adapted from blum et al.
<dig>  <cit>
scanning electron microscopy  of p. lividus was employed to further characterize archenteron cilia and to test a potential polarization of cilia along the animal-vegetal, and thus along the ap axis of the embryo .
embryos, which were broken perpendicularly to the animal-vegetal axis and thus allowed a high-power magnification view inside the archenteron, revealed monocilia of about 4 μm in length .
archenteron cilia were clearly present already in mid gastrula stage embryos .
importantly, using the cilium-insertion point and the cell center as reference points, a clear posterior polarization of cilia was obvious already when cells were orienting towards the inside of the archenteron in animal/anterior direction .
in summary, our descriptive analysis of the archenteron showed that cells harbored monocilia at a time point just prior to the asymmetric induction of nodal, suggesting a functional role homologous to that of vertebrate lros.
the sea urchin gastrula embryo expresses marker genes for motile cilia
next we analyzed the expression of marker genes indicative of motile cilia.
to that end, sea urchin homologs of two marker genes for motile cilia, dynein axonemal heavy chain  <dig>  and forkhead box protein j <dig>  were cloned by rt-pcr and expression patterns during embryonic development were assessed by whole-mount in situ hybridization.
in the ectoderm, dnah <dig> was broadly expressed with intense signals in the apical tuft region.
localized mrna expression was also found in the vegetal part of the gastrula mesendodermal tissue, followed by expression in the archenteron .
sense control probes were negative at all stages examined and for all genes analyzed in this study .
analyses of foxj <dig> mrna expression revealed a similar pattern in the ectoderm and strong staining in the area of the developing apical tuft .
at early gastrula stages, localized expression was found in the vegetal plate region , while in late gastrula stages, a mesodermal expression domain started to appear at the anterior tip of the archenteron .
these expression patterns were indicative of a population of motile mesendodermal cilia in the archenteron and reminiscent of vertebrate lros.fig.
<dig> motile cilia marker genes dnah <dig> and foxj <dig> are expressed throughout sea urchin gastrulation.
whole mount in situ hybridization of early , mid  and late  gastrula stage p. lividus embryos, as well as prism stages  for mrna expression of dnah <dig> , foxj <dig>  and pkd <dig> .
schematic representation of staining in mid-gastrula embryos is highlighted in drawings in .
white arrowheads highlight vegetal blastopore and archenteron tip expression areas.
schematic drawings adapted from blum et al.
<dig>  <cit>
to investigate additional potentially conserved lro genes, we cloned p. lividus bicaudal c homolog  <dig>   <cit> , which is required for cilia polarization at the vertebrate lro, and pkd <dig>  a calcium channel required for sensing of the flow  <cit> .
expression of bicc <dig> has been recently described in the sea urchin hemicentrotus pulcherrimus, i.e.
an unrelated genus.
our analysis in p. lividus confirmed the reported localization, i.e.
strong expression of bicc <dig> mrna at the vegetal pole of early to late gastrula stage embryos, directly at the site of invagination .
p. lividus pkd <dig> mrna  was expressed in a pattern reminiscent of bicc <dig> in early to late gastrula stages, namely in vegetal cells of the early gastrula embryo .
pkd <dig> mrna transcription was further activated in the apical tuft cells and at the archenteron tip of late gastrula to early prism stage embryos .
cilia in the archenteron are motile
in order to directly assess the potential motility of archenteron cilia in live embryos, we analyzed early to mid-gastrula stage embryos by high-speed videography.
to highlight moving objects, movies were processed using a temporal difference imaging method .
additional file 2: movie  <dig> demonstrates that the posteriorly polarized cilia of invaginating archenteron cells  were indeed highly motile.
next we analyzed mid to late gastrula stage embryos, focusing on the lumen of the central part of the elongated archenteron.
again, the monocilia detected by sem analysis  were motile, displaying a rotating pattern .
attempts to visualize a possible effect of cilia motility, i.e.
whether or not this resulted in directed movement of extracellular fluids, failed due to technical reasons, as we were not able to introduce fluorescent micro beads into the archenteron .
despite this shortcoming, our results strongly suggest that the sea urchin embryo harbors a vertebrate-type lro, i.e.
an archenteron epithelium with posteriorly polarized rotating monocilia, which expresses a set of characteristic marker genes, including foxj <dig>  dnah <dig>  bicc <dig> and pkd <dig>
archenteron cilia are required for asymmetric nodal induction
as an alternative to visualizing cilia-driven fluid flow, we chose to directly test the function of cilia in asymmetric nodal induction.
in a first set of experiments we used the pharmacological inhibitor ciliobrevin d, which inhibits the atpase activity of axonemal and cytoplasmic dynein motor proteins  <cit> .
treatment of early gastrula stage embryos with 50 μm ciliobrevin d efficiently inhibited ciliary motility, as judged by direct microscopic observation of embryonic swimming behavior, which completely ceased within minutes .
as this treatment efficiently prevented gastrulation movements as well, we were not able to analyze later nodal expression .
short of a specific inhibitor of axonemal dynein function, we decided to assess the role of cilia for symmetry breakage by removing cilia from the embryo.
to that end, a brief osmotic shock with high-salt  seawater containing twice the normal molarity of nacl  was applied, a procedure previously reported to completely deciliate the ectodermal surface of the embryo  <cit> .
embryos between early gastrula and prism stage were treated with hs seawater for 60– <dig> s and returned to regular seawater until untreated control embryos reached late gastrula to pluteus stages.
embryos were fixed, assessed for developmental defects and/or processed for if, sem or in situ hybridization.
first we analyzed whether this procedure was suited to remove archenteron cilia.
swimming behavior was instantly impaired, as described for ciliobrevin above .
embryos were fixed 20 min after treatment and subjected to if analysis of cilia or sem analysis.
while normal ciliation was seen in sem photographs of untreated embryos, hs-treated specimens were devoid of cilia, both on the outer surface and at the proximal  end of the archenteron cavity .
to investigate whether cilia were removed along the entire archenteron, we analyzed optical sections of treated embryos using if.
control embryos displayed normal ciliation along the extended archenteron.
in contrast, about half of the hs-treated embryos lacked cilia altogether, with the remainder of specimens displaying a small number of very short cilia remnants .
these experiments demonstrated that hs treatment was an efficient tool to remove archenteron cilia from the embryo.fig.
<dig> deciliation impairs lr asymmetric nodal cascade induction.
sem  and if  analyses of cilia in control untreated embryos  and specimens exposed to a 60–90 s osmotic shock .
note that cilia were almost completely absent following high salt treatment.
cilia in  and  were stained with an antibody against anti-acetylated-α-tubulin , nuclei with dapi, and cell boundaries visualized by phalloidin-green.
e, g unilateral induction of asymmetric nodal cascade genes in control embryos.
f, h bilateral ectodermal nodal and pitx <dig> expression following deciliation of early to mid-gastrula stage embryos.
black arrowheads highlight expression in the archenteron and ectoderm; white arrowheads indicate lack of expression.
i quantification of expression pattern from .
unilateral pitx <dig> expression in coelomic pouches of pluteus stage control specimen  and bilateral expression in deciliated embryos .
bilateral expression of pitx <dig> after deciliation is independent of mapk/p <dig> inhibition through sb <dig> .
o quantification of expression patterns.
posterior/vegetal is to the top in  and to the bottom in
because of the proven function of cilia in signal transduction in different species  <cit> , we asked whether deciliation affected normal embryonic development.
the protocol applied here has been previously used to remove ectodermal cilia at different stages of development, without any reports of developmental defects .
stephens et al.
even applied multiple rounds of deciliation  which resulted in phenotypically normal pluteus larvae  <cit> .
in order to confirm that the hs-protocol did not interfere with normal embryogenesis in our experiments as well, > <dig>  embryos were deciliated in  <dig> independent experiments at different time points during development, between late blastula and late gastrula stages.
treated specimens were fixed and assessed for developmental delays or phenotypic alterations at time points when untreated control embryos had reached a) late gastrula with fully elongated archenteron; or b) early pluteus stages.
in both sets of experiments, no phenotypical difference was noted between control and hs-treated samples .
quantitative evaluation of experiments revealed no temporal delay in either of these groups, besides minor and random fluctuations that were sometimes observed .
together with the previously published deciliation approaches, these control experiments demonstrate that the protocol applied here efficiently removed archenteron cilia without impairment of normal development.
in a final series of experiments, we tested whether deciliation impacted on nodal asymmetry in the archenteron and ectoderm, which we hypothesized based on our descriptive analysis of archenteron cilia.
hs treated embryos indeed revealed grossly altered patterns of nodal expression at late gastrula stages, when control embryos displayed unilateral patterns in both, the archenteron and the ectoderm .
surprisingly, hs treatment at early to mid-gastrula stages, i.e.
just before asymmetric induction of the nodal cascade, resulted in an expanded, bilateral ectodermal expression of nodal in the vast majority of treated specimens.
the same result was obtained with pitx <dig> , a direct target of nodal  <cit> .
bilateral activation of the nodal cascade was especially evident when analyzed in the developing coelomic pouches of early pluteus stage embryos, using pitx <dig> as a late lr marker gene .
the archenteron domain of nodal in late gastrula stage embryos, however, was mostly undetectable .
next we tested whether high-salt treatments resulted in a stress-induced activation of mapk/p38-mediated ectopic activation of nodal  <cit> .
if it were, inhibition of mapk/p <dig> should rescue the deciliation-induced aberrant activation of the nodal cascade.
blastula-stage embryos were treated with the mapk/p <dig> pathway-specific inhibitor sb <dig>  <cit> , to test the efficiency of the drug, which resulted in dorso-ventral axis defects .
when embryos were treated after deciliation at mid to late gastrula stages, development was not impaired but specimens still exhibited bilateral pitx <dig> expression in the coelomic pouches at early pluteus stage .
these experiments thus demonstrated that the expanded expression pattern of lr marker genes after deciliation was not due to mapk/p38-mediated over-activation of nodal signaling during gastrulation.
in order to determine whether there was a sensitive time period, i.e.
whether cilia were required only during certain developmental stages, we performed a time course of deciliation and treated specimens with high salt at defined points of development: early gastrula, mid gastrula, late gastrula or prism stage.
based on cilia-driven symmetry breakage in the vertebrates, the time window was expected to be rather narrow.
figure 3i demonstrates that deciliation at very early to mid-gastrula stages, before full extension of the archenteron and before asymmetric nodal expression, caused aberrant lr development.
embryos treated after this point, during late gastrula to early prism stages, showed normal unilateral expression of nodal, i.e.
the sensitive time window closes during late gastrulation.
in summary, our work demonstrates that the sea urchin archenteron harbors polarized and motile monocilia, which are required for lr axis determination during gastrulation.
discussion
theoretical considerations and deductive logics have previously led us to propose that sea urchin embryos possess an ancestral lro, homologous to that of the vertebrates  <cit> .
here we demonstrate in two species that sea urchin gastrula embryos indeed display a mesendodermal monociliated archenteron, which is reminiscent of the vertebrate lro .
importantly, cilia were required for asymmetric nodal induction, arguing for a conserved cilia-based mechanism for lr symmetry breakage in the deuterostome lineage.fig.
<dig> the ciliated left-right organizer: a synapomorphy of the deuterostomes.
a–c schematic drawings of sea urchin , amphioxus  and xenopus  gastrula/neurula stage embryos .
in all cases the archenteron harbors cells of mesodermal fate , which in frog and sea urchin are ciliated and function as lros.
the position of mesodermal lros is indicated by an arrowhead.
d
nodal is evolutionary conserved among metazoans except for the ecdysozoa.
deuterostomes share a ciliated lro as a common synapomorphy.
drawings adapted in parts from blum et al.
<dig>  <cit>
common features between the sea urchin archenteron and vertebrate lros include polarized and motile monocilia as well as expression of dnah <dig> and foxj <dig>  two genes which are mandatory for generation of leftward flow.
although we were not able to visualize a fluid flow within the archenteron directly, we propose that cilia-dependent symmetry breakage is a conserved feature between sea urchin and the vertebrates.
in order to qualify as the nodal inducing event, cilia-driven symmetry breakage should occur at the right time, before asymmetric nodal induction, and in the right place, i.e.
at the archenteron.
besides the presence of motile cilia itself, the expression of vertebrate lro components dnah <dig>  foxj <dig>  and pkd <dig> in this very region are in perfect agreement with this proposal .
not every lro feature seems to be conserved between sea urchins and the vertebrates, though.
while flow-perceiving  cells in vertebrates are located at the posterior pole of the embryo, close to the blastopore or proximal end of the gut, these cells reside at the anterior tip of the archenteron in the sea urchin late gastrula embryo, at its distal end, as deduced from the nodal expression domain .
interestingly, an intermediate scenario is encountered in amphioxus.
here, like in amphibians, a bilateral nodal expression domain was found, but in the anterior part of the archenteron like in sea urchins.
the expression on the right side of amphioxus later disappears, possibly due to a leftward cilia-driven flow  <cit>  .
this reasoning is further supported by the recent description of a dand <dig> homolog, which was found co-expressed with nodal but down-regulated on the left side, resembling the situation at the vertebrate lro  <cit> .
interestingly, a dand <dig> orthologue was not reported for p. lividus, nor was it found in s. purpuratus  <cit> .
considering just two features, localization of the supposed lro and presence or absence of dand <dig>  sea urchins, cephalochordates and vertebrates could represent three distinct states, indicative of evolutionary transitions: while sea urchins and amphioxus share an anterior  position of the lro within the archenteron, amphioxus and vertebrates both possess an orthologue of the nodal antagonist dand <dig>  but only the vertebrates localize their lro close to the blastopore.
what kind of functional adaptations underlie these variations remains to be investigated.
there are additional lr components worth being considered in the evolutionary context, namely pkd <dig> and bicc <dig>  both of which were expressed in early sea urchin gastrula mesendodermal tissues during invagination .
bicc <dig> in mouse and frog functions to polarize lr cilia  <cit> .
in that sense the expression in sea urchins correlated with that in frog and mouse: it is precisely the bicc <dig> expressing cells which harbor cilia polarized to the posterior pole in mid gastrula stage embryos in the sea urchin, and which we here report to be motile .
expression differed in another aspect, however, as bicc <dig> mrna was not colocalized with nodal expression at the archenteron tip, different from the vertebrates, where bicc <dig> is co-expressed with nodal in the lro-flanking cells - and might be involved in sensing of flow  <cit> .
it would be interesting to know where bicc <dig> is expressed in amphioxus, around the blastopore or in the anterior mesoderm.
pkd <dig> in the sea urchin embryo was expressed in the vegetal mesoderm and endoderm at early to mid-gastrula stages and at the tip of the archenteron.
this expression is remarkable, as pkd <dig> has not been reported to be transcriptionally up-regulated in or at any vertebrate lro.
the encoded protein, the calcium channel polycystin- <dig>  however, is present in mouse and fish lros, where it is involved in flow sensing  <cit> .
functional conservation of symmetry breakage is further supported by the expression of pkd <dig> in xenopus gastrula embryos, where it is highly expressed in the mesendodermal ring around the blastopore, reminiscent of the vegetal mesoderm expression in sea urchins .
again, expression in amphioxus has not been reported as yet but should be highly informative.
taken together, the presence of cilia and the expression of conserved lr genes strongly argue for a conserved role of motile archenteron cilia in lr symmetry breakage.
the most convincing result, however, is presented by our analysis of cilia function, where deciliation at a time point just prior to asymmetric induction of nodal in the archenteron resulted in altered expression of the nodal cascade.
molecular asymmetries were lost, with a differential response of nodal and pitx <dig>  while archenteron nodal was mostly absent or below the detection level of the experiment, the ectodermal domain and later on pitx <dig> in the coelomic pouches became expanded and bilateral .
these observations cannot be attributed to the absence of archenteron nodal.
when nodal was experimentally manipulated in the archenteron to be absent or expressed in a bilateral manner, the ectodermal domain remained asymmetrical, although in a randomized manner, being activated either on the left or on the right side  <cit> .
the ectoderm is still competent to express nodal on both sides during gastrulation, as shown by activin treatment of early gastrula embryos  <cit> .
the maintenance of unilateral ectodermal nodal expression might thus be explained by an additional ectodermal cilia function, which was ablated in high salt-treated embryos as well, but we can only speculate on this issue.
our results would be explained if external cilia had a function in restricting nodal cascade activation unilaterally in the ectoderm, and the archenteron cilia provided a biasing cue, as they do during vertebrate lr axis determination  <cit> .
while this manuscript was under review, two papers were published that dealt with symmetry breaking in sea urchin embryos.
in agreement with our conclusions, takemoto et al.
deduced that cilia were required for symmetry breakage in sea urchins.
application of an inhibitor of motile cilia to early blastula stage embryos resulted in a loss of nodal asymmetry, archenteron cilia, however, were not analyzed in this study  <cit> .
warner et al.
injected 1-cell stage embryos with a kinesin- <dig> antibody, which removed all cilia from the time point of injection onwards.
nodal was strongly reduced in the four embryos analyzed, while asymmetric soxe expression in the coelomic pouches was retained in the six embryos included in this one experiment.
the authors concluded that cilia were not required for symmetry breakage but rather for hedgehog signaling-mediated nodal maintenance  <cit> .
we strongly disagree with this conclusion for the following reasons:  lepage and colleagues have shown that asymmetric soxe expression occurs independent of archenteron nodal;  the removal of all cilia from the earliest developmental stages onwards potentially impacts on many more signaling pathways, such as for example hedgehog, as shown by warner et al   <cit> , and thus most probably impact on stages before archenteron cilia emerge;  attenuated nodal expression by permanent cilia ablation, as described by warner et al., might be the result of a) loss of archenteron cilia function, which causes bilateral expanded nodal expression as shown in our present work, followed in time by b) loss of hedgehog-mediated nodal maintenance, as described by warner et al.
.
we like to extend our evolutionary considerations to the precursor tissue of the lro in the sea urchin.
keller and colleagues previously revitalized the concept of the superficial mesoderm  in the chordate lineage.
best characterized in amphibians, the sm of the pre-gastrula embryo gives rise to the archenteron lro during gastrula/neurula stages to end up in the axial and paraxial mesoderm during later embryogenesis  <cit> .
besides the birds, which lack an lro, the sm has been identified in most chordate lineages  <cit>  .
in the sea urchin mesenchyme blastula, the non-skeletogenic mesoderm, together with the endoderm, locates superficially at the vegetal pole as well, reminiscent of the situation in amphibians  <cit> .
of the known sm marker genes in the frog, foxj <dig> , nodal <dig>  <cit>  and wnt11b  <cit> , only foxj <dig> has been analyzed in sea urchins .
its mrna localization in the early gastrula vegetal cells indeed argues for a conserved blastula stage sm .
furthermore, like in vertebrate sm tissues, these cells show accumulation of nuclear ß-catenin at the very time point when the sm is specified  <cit> .
this is of relevance, as foxj <dig> expression has been shown to be directly induced by canonical wnt signaling.
CONCLUSIONS
we conclude that the early sea urchin embryo represents an ancestral deuterostome state with a vegetal sm at blastula stages, which transforms into an archenteron lro, where motile cilia are necessary to break the bilateral symmetry.
cilia-driven symmetry breakage thus should represent a synapomorphy of the deuterostome lineage.

 vertebrates possess pronounced visceral asymmetries along their left-right body axis, although they belong to the large phylogenetic group of the bilateria, which refers to their bilaterally symmetric outer appearance. most organs are positioned in a characteristic way in the thoracic and abdominal cavities. in all vertebrate species examined so far, these asymmetries are under the control of the nodal signaling cascade, which is only activated in the left lateral plate mesoderm before the first appearance of anatomical asymmetries. the secreted transforming growth factor beta nodal binds to its receptor which results in the activation of its own transcription and that of its negative feedback inhibitor lefty, another secreted tgfβ superfamily member. lefty antagonizes nodal signaling, providing its temporal and spatial control. additionally, the paired-like homeodomain transcription factor pitx is induced downstream of nodal and mediates, through less well-known target gene activation, the setup of asymmetric organ morphogenesis. the event activating this highly conserved developmental program is referred to as symmetry breakage. even though variations of the common theme may exist, an ancestral mode of vertebrate symmetry breaking has emerged over the past few years: at the heart of this mechanism acts an extracellular leftward fluid flow, generated by a transient ciliated epithelium, the so-called left-right organizer. the vertebrate lro in amphibians, and posterior notochord or ventral node in mammals) represents a field of mono-ciliated cells at the posterior end of the forming notochord, flanked by endodermal archenteron cells. this unique tissue, which develops during early neurula stages, consists of superficially located mesendodermal cells which are transiently placed in the primitive gut or archenteron, where they function as the lro. the cells of the lro subsequently leave the epithelium to ingress into deeper mesodermal structures. the polarized attachment of cilia at the posterior cell surface, together with their clockwise rotational movement, create an asymmetric stimulus by the leftward acceleration of extracellular fluids, i.e. a leftward flow. this setup has been functionally described in mammals, amphibians and teleost fish, and homologous tissues have been identified in salamanders and basal bony fish. interference with ciliary length, motility, polarization or flow function in general resulted in defects of the lr axis. as a result of leftward flow, the nodal inhibitor dand becomes down-regulated on the left margin of the lro, where it is co-expressed on both sides with nodal itself. nodal thus becomes liberated on the left side to induce the asymmetric signaling cascade in the left lateral plate mesoderm. dand repression is induced through a flow-dependent intracellular calcium signal, which is mediated through the calcium channel pkd. although ubiquitously expressed during these stages, pkd inhibition causes lr defects and prevents a unilateral calcium signal in mouse and fish lros. outside of the vertebrate lineage, lr asymmetries are common as well. for many deuterostome lineages, unilateral nodal expression has been described, which seems to be the common mediator of lr asymmetries in metazoan animals. tunicates, the sister group of the vertebrates, as well as the cephalochordates, the most basal group of chordates, both express nodal asymmetrically on the left side. the latter seem to show an ancient state of this set-up, with many lro targets activated similarly to vertebrates, such as nodal, lefty, pitx and dand. accordingly, interfering with nodal activity in the cephalochordate amphioxus also resulted in lr defects. the existence of an lro in amphioxus has been predicted, but not yet analyzed or functionally tested. within the deuterostomes, the ambulacraria, a chordate sister group which comprises the echinoderms, show unilateral nodal activation during late gastrula stages as well. although adult echinoderms took the path of reestablishing a radially pentameric body plan during evolution, their early embryo displays the typical bilaterally symmetrical embryonic development, which results in a pronounced lr asymmetry before metamorphosis. as in other deuterostomes, nodal is the first asymmetrically activated gene, and is first found on one side in the developing archenteron tip. the functional requirement and a complex downstream gene regulatory network have been elucidated in detail in sea urchin embryos. this asymmetric archenteron domain instructs a second symmetrical ectodermal nodal domain to switch towards this side as well. this expression is thought to be on the right, as deduced from a ventral mouth opening. we have previously argued that this difference can be resolved to a common evolutionary origin, if the ventral side of echinoderms is homologized with the dorsal side of chordates. this reasoning is based on the expression of dorsal organizer genes in the oral ectoderm of the sea urchin larva, suggesting that the mouth - counterintuitively - opens on the dorsal side. such an evolutionary mouth repositioning can be easily envisaged as a transitional process from echinoderm ancestors to vertebrates. indeed, oral organizer identity has been recently shown to be functionally conserved in the sea urchin p. lividus. thus, in this scenario, left-asymmetric nodal expression is a synapomorphy of the deuterostomes. one major question, however, has remained unanswered: how does symmetry breakage upstream of asymmetric nodal induction occur in echinoderms? do sea urchin embryos possess a lro or an evolutionary functional precursor that induces asymmetric nodal expression? do archenteron cells possess cilia and, if so, are these required to induce asymmetric nodal expression? using descriptive and functional approaches we show that archenteron cells in the sea urchin larva harbor monocilia; archenteron cilia are polarized and motile; and cilia are required for asymmetric nodal induction.  sea urchin archenteron cells harbor polarized monocilia the defining feature of vertebrate lros are polarized monocilia. as an entry point into studying sea urchin symmetry breakage, we investigated the presence of cilia on staged gastrula embryos by performing immunofluorescence with a well-characterized anti-acetylated α-tubulin antibody. in addition to the previously described long ectodermal cilia, optical sections of gastrula stage paracentrotus lividus embryos revealed a population of shorter cilia within the developing archenteron. higher magnification showed that most cilia ranged from 4–6 μm in length, with the most anterior ones being longer, measuring up to 10 μm. the same basic characteristics of mesendodermal archenteron cilia were also found in a second sea urchin species, strongylocentrotus pallidus, demonstrating the conserved presence of cilia in the primitive gut of sea urchin larvae. one slight variation was observed, namely that very early gastrula embryos of strongylocentrotus apparently lacked archenteron cilia, which were, however, present at mid to late gastrula stages. in p. lividus, cilia were present already at earlier gastrula stages.fig. polarized cilia at the sea urchin archenteron. a–e p. lividus and s. pallidus embryos were analyzed by if for the presence of cilia at the archenteron. optical sections showed ectodermal and archenteron cilia at mid gastrula stages. late but not early s. pallidus gastrula stage embryos revealed cilia in the archenteron and at the archenteron tip. cilia were stained with an antibody against acetylated-α-tubulin or anti-α-tubulin, nuclei were stained with dapi, and cell boundaries were visualized by phalloidin-green or phalloidin-red. f, g sem analysis of p. lividus ectodermal and archenteron cilia. fractured embryos allowed the visualization of monocilia on archenteron cells. cilia are highlighted by an arrowhead posterior polarization of cilia on cells which invaginated into the archenteron. cilia are colored in yellow and individual cells alternating in green and purple. the ciliary base is marked by a red semicircle, the center of the cell is indicated by a yellow dot. schematic drawings adapted from blum et al. scanning electron microscopy of p. lividus was employed to further characterize archenteron cilia and to test a potential polarization of cilia along the animal-vegetal, and thus along the ap axis of the embryo. embryos, which were broken perpendicularly to the animal-vegetal axis and thus allowed a high-power magnification view inside the archenteron, revealed monocilia of about 4 μm in length. archenteron cilia were clearly present already in mid gastrula stage embryos. importantly, using the cilium-insertion point and the cell center as reference points, a clear posterior polarization of cilia was obvious already when cells were orienting towards the inside of the archenteron in animal/anterior direction. in summary, our descriptive analysis of the archenteron showed that cells harbored monocilia at a time point just prior to the asymmetric induction of nodal, suggesting a functional role homologous to that of vertebrate lros. the sea urchin gastrula embryo expresses marker genes for motile cilia next we analyzed the expression of marker genes indicative of motile cilia. to that end, sea urchin homologs of two marker genes for motile cilia, dynein axonemal heavy chain and forkhead box protein j were cloned by rt-pcr and expression patterns during embryonic development were assessed by whole-mount in situ hybridization. in the ectoderm, dnah was broadly expressed with intense signals in the apical tuft region. localized mrna expression was also found in the vegetal part of the gastrula mesendodermal tissue, followed by expression in the archenteron. sense control probes were negative at all stages examined and for all genes analyzed in this study. analyses of foxj mrna expression revealed a similar pattern in the ectoderm and strong staining in the area of the developing apical tuft. at early gastrula stages, localized expression was found in the vegetal plate region, while in late gastrula stages, a mesodermal expression domain started to appear at the anterior tip of the archenteron. these expression patterns were indicative of a population of motile mesendodermal cilia in the archenteron and reminiscent of vertebrate lros.fig. motile cilia marker genes dnah and foxj are expressed throughout sea urchin gastrulation. whole mount in situ hybridization of early, mid and late gastrula stage p. lividus embryos, as well as prism stages for mrna expression of dnah, foxj and pkd. schematic representation of staining in mid-gastrula embryos is highlighted in drawings in. white arrowheads highlight vegetal blastopore and archenteron tip expression areas. schematic drawings adapted from blum et al. to investigate additional potentially conserved lro genes, we cloned p. lividus bicaudal c homolog, which is required for cilia polarization at the vertebrate lro, and pkd a calcium channel required for sensing of the flow. expression of bicc has been recently described in the sea urchin hemicentrotus pulcherrimus, i.e. an unrelated genus. our analysis in p. lividus confirmed the reported localization, i.e. strong expression of bicc mrna at the vegetal pole of early to late gastrula stage embryos, directly at the site of invagination. p. lividus pkd mrna was expressed in a pattern reminiscent of bicc in early to late gastrula stages, namely in vegetal cells of the early gastrula embryo. pkd mrna transcription was further activated in the apical tuft cells and at the archenteron tip of late gastrula to early prism stage embryos. cilia in the archenteron are motile in order to directly assess the potential motility of archenteron cilia in live embryos, we analyzed early to mid-gastrula stage embryos by high-speed videography. to highlight moving objects, movies were processed using a temporal difference imaging method. additional file 2: movie demonstrates that the posteriorly polarized cilia of invaginating archenteron cells were indeed highly motile. next we analyzed mid to late gastrula stage embryos, focusing on the lumen of the central part of the elongated archenteron. again, the monocilia detected by sem analysis were motile, displaying a rotating pattern. attempts to visualize a possible effect of cilia motility, i.e. whether or not this resulted in directed movement of extracellular fluids, failed due to technical reasons, as we were not able to introduce fluorescent micro beads into the archenteron. despite this shortcoming, our results strongly suggest that the sea urchin embryo harbors a vertebrate-type lro, i.e. an archenteron epithelium with posteriorly polarized rotating monocilia, which expresses a set of characteristic marker genes, including foxj dnah bicc and pkd archenteron cilia are required for asymmetric nodal induction as an alternative to visualizing cilia-driven fluid flow, we chose to directly test the function of cilia in asymmetric nodal induction. in a first set of experiments we used the pharmacological inhibitor ciliobrevin d, which inhibits the atpase activity of axonemal and cytoplasmic dynein motor proteins. treatment of early gastrula stage embryos with 50 μm ciliobrevin d efficiently inhibited ciliary motility, as judged by direct microscopic observation of embryonic swimming behavior, which completely ceased within minutes. as this treatment efficiently prevented gastrulation movements as well, we were not able to analyze later nodal expression. short of a specific inhibitor of axonemal dynein function, we decided to assess the role of cilia for symmetry breakage by removing cilia from the embryo. to that end, a brief osmotic shock with high-salt seawater containing twice the normal molarity of nacl was applied, a procedure previously reported to completely deciliate the ectodermal surface of the embryo. embryos between early gastrula and prism stage were treated with hs seawater for 60– s and returned to regular seawater until untreated control embryos reached late gastrula to pluteus stages. embryos were fixed, assessed for developmental defects and/or processed for if, sem or in situ hybridization. first we analyzed whether this procedure was suited to remove archenteron cilia. swimming behavior was instantly impaired, as described for ciliobrevin above. embryos were fixed 20 min after treatment and subjected to if analysis of cilia or sem analysis. while normal ciliation was seen in sem photographs of untreated embryos, hs-treated specimens were devoid of cilia, both on the outer surface and at the proximal end of the archenteron cavity. to investigate whether cilia were removed along the entire archenteron, we analyzed optical sections of treated embryos using if. control embryos displayed normal ciliation along the extended archenteron. in contrast, about half of the hs-treated embryos lacked cilia altogether, with the remainder of specimens displaying a small number of very short cilia remnants. these experiments demonstrated that hs treatment was an efficient tool to remove archenteron cilia from the embryo.fig. deciliation impairs lr asymmetric nodal cascade induction. sem and if analyses of cilia in control untreated embryos and specimens exposed to a 60–90 s osmotic shock. note that cilia were almost completely absent following high salt treatment. cilia in and were stained with an antibody against anti-acetylated-α-tubulin, nuclei with dapi, and cell boundaries visualized by phalloidin-green. e, g unilateral induction of asymmetric nodal cascade genes in control embryos. f, h bilateral ectodermal nodal and pitx expression following deciliation of early to mid-gastrula stage embryos. black arrowheads highlight expression in the archenteron and ectoderm; white arrowheads indicate lack of expression. i quantification of expression pattern from. unilateral pitx expression in coelomic pouches of pluteus stage control specimen and bilateral expression in deciliated embryos. bilateral expression of pitx after deciliation is independent of mapk/p inhibition through sb. o quantification of expression patterns. posterior/vegetal is to the top in and to the bottom in because of the proven function of cilia in signal transduction in different species, we asked whether deciliation affected normal embryonic development. the protocol applied here has been previously used to remove ectodermal cilia at different stages of development, without any reports of developmental defects. stephens et al. even applied multiple rounds of deciliation which resulted in phenotypically normal pluteus larvae. in order to confirm that the hs-protocol did not interfere with normal embryogenesis in our experiments as well, > embryos were deciliated in independent experiments at different time points during development, between late blastula and late gastrula stages. treated specimens were fixed and assessed for developmental delays or phenotypic alterations at time points when untreated control embryos had reached a) late gastrula with fully elongated archenteron; or b) early pluteus stages. in both sets of experiments, no phenotypical difference was noted between control and hs-treated samples. quantitative evaluation of experiments revealed no temporal delay in either of these groups, besides minor and random fluctuations that were sometimes observed. together with the previously published deciliation approaches, these control experiments demonstrate that the protocol applied here efficiently removed archenteron cilia without impairment of normal development. in a final series of experiments, we tested whether deciliation impacted on nodal asymmetry in the archenteron and ectoderm, which we hypothesized based on our descriptive analysis of archenteron cilia. hs treated embryos indeed revealed grossly altered patterns of nodal expression at late gastrula stages, when control embryos displayed unilateral patterns in both, the archenteron and the ectoderm. surprisingly, hs treatment at early to mid-gastrula stages, i.e. just before asymmetric induction of the nodal cascade, resulted in an expanded, bilateral ectodermal expression of nodal in the vast majority of treated specimens. the same result was obtained with pitx, a direct target of nodal. bilateral activation of the nodal cascade was especially evident when analyzed in the developing coelomic pouches of early pluteus stage embryos, using pitx as a late lr marker gene. the archenteron domain of nodal in late gastrula stage embryos, however, was mostly undetectable. next we tested whether high-salt treatments resulted in a stress-induced activation of mapk/p38-mediated ectopic activation of nodal. if it were, inhibition of mapk/p should rescue the deciliation-induced aberrant activation of the nodal cascade. blastula-stage embryos were treated with the mapk/p pathway-specific inhibitor sb, to test the efficiency of the drug, which resulted in dorso-ventral axis defects. when embryos were treated after deciliation at mid to late gastrula stages, development was not impaired but specimens still exhibited bilateral pitx expression in the coelomic pouches at early pluteus stage. these experiments thus demonstrated that the expanded expression pattern of lr marker genes after deciliation was not due to mapk/p38-mediated over-activation of nodal signaling during gastrulation. in order to determine whether there was a sensitive time period, i.e. whether cilia were required only during certain developmental stages, we performed a time course of deciliation and treated specimens with high salt at defined points of development: early gastrula, mid gastrula, late gastrula or prism stage. based on cilia-driven symmetry breakage in the vertebrates, the time window was expected to be rather narrow. figure 3i demonstrates that deciliation at very early to mid-gastrula stages, before full extension of the archenteron and before asymmetric nodal expression, caused aberrant lr development. embryos treated after this point, during late gastrula to early prism stages, showed normal unilateral expression of nodal, i.e. the sensitive time window closes during late gastrulation. in summary, our work demonstrates that the sea urchin archenteron harbors polarized and motile monocilia, which are required for lr axis determination during gastrulation. discussion theoretical considerations and deductive logics have previously led us to propose that sea urchin embryos possess an ancestral lro, homologous to that of the vertebrates. here we demonstrate in two species that sea urchin gastrula embryos indeed display a mesendodermal monociliated archenteron, which is reminiscent of the vertebrate lro. importantly, cilia were required for asymmetric nodal induction, arguing for a conserved cilia-based mechanism for lr symmetry breakage in the deuterostome lineage.fig. the ciliated left-right organizer: a synapomorphy of the deuterostomes. a–c schematic drawings of sea urchin, amphioxus and xenopus gastrula/neurula stage embryos. in all cases the archenteron harbors cells of mesodermal fate, which in frog and sea urchin are ciliated and function as lros. the position of mesodermal lros is indicated by an arrowhead. d nodal is evolutionary conserved among metazoans except for the ecdysozoa. deuterostomes share a ciliated lro as a common synapomorphy. drawings adapted in parts from blum et al. common features between the sea urchin archenteron and vertebrate lros include polarized and motile monocilia as well as expression of dnah and foxj two genes which are mandatory for generation of leftward flow. although we were not able to visualize a fluid flow within the archenteron directly, we propose that cilia-dependent symmetry breakage is a conserved feature between sea urchin and the vertebrates. in order to qualify as the nodal inducing event, cilia-driven symmetry breakage should occur at the right time, before asymmetric nodal induction, and in the right place, i.e. at the archenteron. besides the presence of motile cilia itself, the expression of vertebrate lro components dnah foxj and pkd in this very region are in perfect agreement with this proposal. not every lro feature seems to be conserved between sea urchins and the vertebrates, though. while flow-perceiving cells in vertebrates are located at the posterior pole of the embryo, close to the blastopore or proximal end of the gut, these cells reside at the anterior tip of the archenteron in the sea urchin late gastrula embryo, at its distal end, as deduced from the nodal expression domain. interestingly, an intermediate scenario is encountered in amphioxus. here, like in amphibians, a bilateral nodal expression domain was found, but in the anterior part of the archenteron like in sea urchins. the expression on the right side of amphioxus later disappears, possibly due to a leftward cilia-driven flow. this reasoning is further supported by the recent description of a dand homolog, which was found co-expressed with nodal but down-regulated on the left side, resembling the situation at the vertebrate lro. interestingly, a dand orthologue was not reported for p. lividus, nor was it found in s. purpuratus. considering just two features, localization of the supposed lro and presence or absence of dand sea urchins, cephalochordates and vertebrates could represent three distinct states, indicative of evolutionary transitions: while sea urchins and amphioxus share an anterior position of the lro within the archenteron, amphioxus and vertebrates both possess an orthologue of the nodal antagonist dand but only the vertebrates localize their lro close to the blastopore. what kind of functional adaptations underlie these variations remains to be investigated. there are additional lr components worth being considered in the evolutionary context, namely pkd and bicc both of which were expressed in early sea urchin gastrula mesendodermal tissues during invagination. bicc in mouse and frog functions to polarize lr cilia. in that sense the expression in sea urchins correlated with that in frog and mouse: it is precisely the bicc expressing cells which harbor cilia polarized to the posterior pole in mid gastrula stage embryos in the sea urchin, and which we here report to be motile. expression differed in another aspect, however, as bicc mrna was not colocalized with nodal expression at the archenteron tip, different from the vertebrates, where bicc is co-expressed with nodal in the lro-flanking cells - and might be involved in sensing of flow. it would be interesting to know where bicc is expressed in amphioxus, around the blastopore or in the anterior mesoderm. pkd in the sea urchin embryo was expressed in the vegetal mesoderm and endoderm at early to mid-gastrula stages and at the tip of the archenteron. this expression is remarkable, as pkd has not been reported to be transcriptionally up-regulated in or at any vertebrate lro. the encoded protein, the calcium channel polycystin- however, is present in mouse and fish lros, where it is involved in flow sensing. functional conservation of symmetry breakage is further supported by the expression of pkd in xenopus gastrula embryos, where it is highly expressed in the mesendodermal ring around the blastopore, reminiscent of the vegetal mesoderm expression in sea urchins. again, expression in amphioxus has not been reported as yet but should be highly informative. taken together, the presence of cilia and the expression of conserved lr genes strongly argue for a conserved role of motile archenteron cilia in lr symmetry breakage. the most convincing result, however, is presented by our analysis of cilia function, where deciliation at a time point just prior to asymmetric induction of nodal in the archenteron resulted in altered expression of the nodal cascade. molecular asymmetries were lost, with a differential response of nodal and pitx while archenteron nodal was mostly absent or below the detection level of the experiment, the ectodermal domain and later on pitx in the coelomic pouches became expanded and bilateral. these observations cannot be attributed to the absence of archenteron nodal. when nodal was experimentally manipulated in the archenteron to be absent or expressed in a bilateral manner, the ectodermal domain remained asymmetrical, although in a randomized manner, being activated either on the left or on the right side. the ectoderm is still competent to express nodal on both sides during gastrulation, as shown by activin treatment of early gastrula embryos. the maintenance of unilateral ectodermal nodal expression might thus be explained by an additional ectodermal cilia function, which was ablated in high salt-treated embryos as well, but we can only speculate on this issue. our results would be explained if external cilia had a function in restricting nodal cascade activation unilaterally in the ectoderm, and the archenteron cilia provided a biasing cue, as they do during vertebrate lr axis determination. while this manuscript was under review, two papers were published that dealt with symmetry breaking in sea urchin embryos. in agreement with our conclusions, takemoto et al. deduced that cilia were required for symmetry breakage in sea urchins. application of an inhibitor of motile cilia to early blastula stage embryos resulted in a loss of nodal asymmetry, archenteron cilia, however, were not analyzed in this study. warner et al. injected 1-cell stage embryos with a kinesin- antibody, which removed all cilia from the time point of injection onwards. nodal was strongly reduced in the four embryos analyzed, while asymmetric soxe expression in the coelomic pouches was retained in the six embryos included in this one experiment. the authors concluded that cilia were not required for symmetry breakage but rather for hedgehog signaling-mediated nodal maintenance. we strongly disagree with this conclusion for the following reasons: lepage and colleagues have shown that asymmetric soxe expression occurs independent of archenteron nodal; the removal of all cilia from the earliest developmental stages onwards potentially impacts on many more signaling pathways, such as for example hedgehog, as shown by warner et al, and thus most probably impact on stages before archenteron cilia emerge; attenuated nodal expression by permanent cilia ablation, as described by warner et al., might be the result of a) loss of archenteron cilia function, which causes bilateral expanded nodal expression as shown in our present work, followed in time by b) loss of hedgehog-mediated nodal maintenance, as described by warner et al.. we like to extend our evolutionary considerations to the precursor tissue of the lro in the sea urchin. keller and colleagues previously revitalized the concept of the superficial mesoderm in the chordate lineage. best characterized in amphibians, the sm of the pre-gastrula embryo gives rise to the archenteron lro during gastrula/neurula stages to end up in the axial and paraxial mesoderm during later embryogenesis. besides the birds, which lack an lro, the sm has been identified in most chordate lineages. in the sea urchin mesenchyme blastula, the non-skeletogenic mesoderm, together with the endoderm, locates superficially at the vegetal pole as well, reminiscent of the situation in amphibians. of the known sm marker genes in the frog, foxj, nodal and wnt11b, only foxj has been analyzed in sea urchins. its mrna localization in the early gastrula vegetal cells indeed argues for a conserved blastula stage sm. furthermore, like in vertebrate sm tissues, these cells show accumulation of nuclear ß-catenin at the very time point when the sm is specified. this is of relevance, as foxj expression has been shown to be directly induced by canonical wnt signaling.  we conclude that the early sea urchin embryo represents an ancestral deuterostome state with a vegetal sm at blastula stages, which transforms into an archenteron lro, where motile cilia are necessary to break the bilateral symmetry. cilia-driven symmetry breakage thus should represent a synapomorphy of the deuterostome lineage. 
based on these results we argue that the anterior archenteron represents a bona fide lro and propose that cilia-based symmetry breakage is a synapomorphy of the deuterostomes. sea urchindeuterostomesleft-right asymmetrysymmetry breakagenodalcilialeft-right organizerhttp://dx.doi.org/ /501100001659deutsche forschungsgemeinschaftbl285/9-2blum martin http://dx.doi.org/ /501100001659deutsche forschungsgemeinschaftbl285/10-1blum martin http://dx.doi.org/ /501100002261russian foundation for basic research14-04- mol_akremnyov stanislav http://dx.doi.org/ /501100002347bundesministerium für bildung und forschung01pl humboldt reloadedtisler matthias issue-copyright-statement© the author 2016 in most vertebrates, asymmetric nodal induction results from a cilia-driven leftward fluid flow at the left-right organizer, a ciliated epithelium present during gastrula/neurula stages. high-speed videography revealed rotating cilia in the archenteron slightly before asymmetric nodal induction. our data demonstrate that cilia - like in vertebrates - are required for asymmetric nodal induction in sea urchin embryos. here we study sea urchin embryos, which use nodal to establish larval lr asymmetry as well. expression of foxj and dnah suggested that archenteron cilia were motile. cilia were polarized to the posterior pole of cells, a prerequisite of directed flow. left-right organ asymmetries are a common feature of metazoan animals. removal of cilia through brief high salt treatments resulted in aberrant patterns of nodal expression.
 left-right organ asymmetries are a common feature of metazoan animals. in many cases, laterality is established by a conserved asymmetric nodal signaling cascade during embryogenesis. in most vertebrates, asymmetric nodal induction results from a cilia-driven leftward fluid flow at the left-right organizer, a ciliated epithelium present during gastrula/neurula stages. conservation of lro and flow beyond the vertebrates has not been reported yet.  here we study sea urchin embryos, which use nodal to establish larval lr asymmetry as well. cilia were found in the archenteron of embryos undergoing gastrulation. expression of foxj and dnah suggested that archenteron cilia were motile. cilia were polarized to the posterior pole of cells, a prerequisite of directed flow. high-speed videography revealed rotating cilia in the archenteron slightly before asymmetric nodal induction. removal of cilia through brief high salt treatments resulted in aberrant patterns of nodal expression. our data demonstrate that cilia - like in vertebrates - are required for asymmetric nodal induction in sea urchin embryos.  based on these results we argue that the anterior archenteron represents a bona fide lro and propose that cilia-based symmetry breakage is a synapomorphy of the deuterostomes. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords sea urchindeuterostomesleft-right asymmetrysymmetry breakagenodalcilialeft-right organizerhttp://dx.doi.org/ /501100001659deutsche forschungsgemeinschaftbl285/9-2blum martin http://dx.doi.org/ /501100001659deutsche forschungsgemeinschaftbl285/10-1blum martin http://dx.doi.org/ /501100002261russian foundation for basic research14-04- mol_akremnyov stanislav http://dx.doi.org/ /501100002347bundesministerium für bildung und forschung01pl humboldt reloadedtisler matthias issue-copyright-statement© the author 2016 