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Seminars in Cell & Developmental Biology 20 (2009) 464–471

Contents lists available at ScienceDirect

Seminars in Cell & Developmental Biology

journa l homepage: www.e lsev ier .com/ locate /semcdb

eview

volution of leftward flow

artin Blum ∗, Thomas Weber, Tina Beyer, Philipp Vickniversity of Hohenheim, Institute of Zoology, Garbenstrasse 30, 70593 Stuttgart, Germany

r t i c l e i n f o

rticle history:

a b s t r a c t

The asymmetric Nodal signaling cascade as a prerequisite for asymmetric body plan specification is con-

vailable online 14 November 2008

eywords:eft-right asymmetryvolutionodal

served among deuterostomes. In this review we argue that symmetry breakage by cilia-driven leftwardflow presents an ancestral character of vertebrates, likely the chordate phylum and maybe all deuteros-tomes. In vertebrates, leftward flow occurs in a transient structure, a monociliated epithelium, which isderived from superficial mesoderm and localizes to the archenteron roof during gastrulation. The chickas an example for the highly derived birds lacks superficial mesoderm and flow. This loss should besecondary, as flow is present from fish and amphibians to mammals.

iliauperficial mesoderm © 2008 Elsevier Ltd. All rights reserved.

ontents

1. Flow in vertebrate embryos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4651.1. Mammals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4651.2. Amphibians . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4661.3. Fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467

1.3.1. Teleost fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4671.3.2. Chondrostean fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467

1.4. Common denominator of vertebrate flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4671.5. Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468

2. The chick problem. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468

3. Primitive chordates: predictions for amphioxus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4684. Deuterostomes: speculations about sea urchins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4695. Conclusion/outlook. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469

. . . . .. . . . .

nd intestines). In addition, the brain displays functional and/ororphological asymmetries such as the location of speech cen-

ers in humans or the development of the habenulae in teleost fish6,7]. Outside the vertebrates, other types of asymmetries are found.n the lower chordate amphioxus, a great number of asymmetries

∗ Corresponding author. Tel.: +49 711 4592 2255; fax: +49 711 4592 3450.E-mail address: mblum@uni-hohenheim.de (M. Blum).

084-9521/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.oi:10.1016/j.semcdb.2008.11.005

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were reported [4,8,9]. Among others, mouth and anus form on theleft side, and the somites develop asymmetrically. In echinoderms,the right coelomic pouch of the larva degenerates, while most adulttissues develop from the left side [3,10,11].

Despite the conserved nature of the Nodal signaling cascade,mechanisms underlying the initial asymmetric induction of Nodalhave been thought to differ greatly even among the vertebrates[12,13]. A cilia-based flow of extracellular fluid from right to leftwas described in mammals (mouse [14] and rabbit [15]), teleostfish (zebrafish [16,17] and medaka [15]), and – recently – the frogXenopus laevis [18]. Absence or disturbance of flow in all casesinduced laterality defects. Early asymmetries in Xenopus and chick,

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

All deuterostomes looked at to date possess an asymmetricNodal signaling cascade, which acts at gastrula/neurula stagesof development [1–5]. Asymmetric Nodal signaling precedesasymmetric morphogenesis, which comes in different flavors. Invertebrates, many organs adopt asymmetric positions in the tho-racic and abdominal cavities (i.e. heart, lung, stomach, liver, spleen,

and absence of flow in chick embryos, have questioned flow as theuniversal vertebrate mechanism of symmetry breakage [13,19–22].

In the present review we argue that cilia-driven vectorial flowrepresents the ancestral mode of symmetry breakage in vertebrates,

M. Blum et al. / Seminars in Cell & Developmental Biology 20 (2009) 464–471 465

Fig. 1. Sites of leftward flow in the vertebrates. (A and B) Mammals. PNC in a 3-somite rabbit (A) and an E8.0 mouse embryo (B). (C–E) Amphibians. GRP in neurula embryoso e 17/1s . Dashh wo moB ier. Pa

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f Xenopus laevis (C; stage 18), Xenopus tropicalis (D; stage 15/16) and axolotl (E; stagtage 25). SEM pictures of embryos shown in ventral view and oriented anterior upigher power magnifications of ciliated cells. Red arrowheads in inset of (G) mark t′ , F′ , insets in C–E, G). (Panels F + F′ reprinted from [15] with permission from Elsev

r even chordates. In addition we reason that flow may have beenresent already in a common ancestor of the deuterostomes.

. Flow in vertebrate embryos

.1. Mammals

An involvement of ciliary motility in laterality determination haseen known since 1976, when Afzelius reported that Kartageneryndrome, a human disease characterized – among other pheno-ypes – by randomized organ situs, was caused by immotile cilia23]. Schoenwolf and coworkers in a seminal paper in 1994 on node

8). (F and G) Fish. KV in medaka (F) and GRP in the white sturgeon (G, stage 21; G′ ,ed lines highlight ciliated epithelia. (A′), (B′), (F′) and insets in (C–E) and (G) shownocilia. Scale bars represent 50 �m (A–D, F, G, and G′), 200 �m (E), and 10 �m (A′ ,

nel G reprinted from [42] with permission from Wiley.)

and notochord morphogenesis first described monocilia on thesecells, and in a bold statement speculated that these cilia might beinvolved in the establishment of sidedness [24]. Cilia-driven left-ward flow was discovered in 1998 by Hirokawa and coworkers [14].Their elegant work demonstrated that mouse embryos have motilemonocilia at a structure commonly referred to as ‘node’ (but seeSection 1.5 below), and that these cilia rotate in a clockwise fashion

to produce a directed fluid flow from right to left in the extracellularspace [14]. Knockout mouse embryos mutant for the motor proteinKif3A lacked cilia and displayed laterality defects [14]. Meanwhilea multitude of factors have been described which are involved inciliogenesis or ciliary motility, mutations of which invariably result

466 M. Blum et al. / Seminars in Cell & Developmental Biology 20 (2009) 464–471

Fig. 2. Localization and size of KV, GRP and PNC. SEM pictures of medaka larva (A), posterior half of X. laevis neurula (B), rabbit blastodisc (C) and mouse egg cylinder (D),s dorsas knows D), 20E

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hown in ventral view and oriented anterior up in (A, C and D), or in anterior viewhape of ciliated field of cells in various embryos. Cilia numbers are indicated where, somite. Red arrowheads mark organizer tissue. Scale bars represent 50 �m (A +lsevier.)

n left-right (LR) defects [2,12,13,25]. Probably the most convinc-ng demonstration of the decisive role of leftward flow in lateralitypecification was provided by Hamada and coworkers who invertedhe situs by artificial rightward flow applied to wildtype embryosn culture, and rescued mutant embryos with immotile cilia bypplying an artificial flow to the left [26].

In a detailed morphological and histological study we have ana-yzed the node region in the rabbit embryo [27], the model organismn which Hensen first described the node in 1876 [28]. No cilia wereound on Hensen’s node, however, monocilia reminiscent of thenes in the mouse were discovered all along the notochordal plate[27]; Fig. 1A and A′). Indeed, these cilia rotate in a clockwise fash-on and produce a leftward flow in the wide posterior portion ofhe notochordal plate (posterior notochord; PNC), just like in mouse[15] and our unpublished results). The rabbit embryo develops viaflat blastodisc ([29]; Fig. 2C), such as most mammalian embryosut in contrast to the mouse, which presents itself as cup-shapedtructure (egg cylinder; Fig. 2D) at gastrula/neurula stages, withhe ventral surface facing outward. Based on ciliation and markerene expression we have previously argued that the ciliated dis-al indentation of the egg cylinder is homologous to the rabbit PNCnd distinct from the node, i.e. the homolog of Spemann’s organizer[27]; cf. Fig. 1A, B and A′, B′).

Thus, in the two mammalian species analyzed to date a field ofotile monocilia emerges during late gastrula/early neurula stages.

ilia rotate in a clockwise fashion, and due to their posterior tiltroduce a leftward fluid flow [30,31]. Initially it was thought thatR monocilia were exclusively of the 9 + 0 axoneme type [14,24].ur analysis of PNC cilia in rabbit revealed three types of axonemeshich were present in a salt-and-pepper pattern, namely 9 + 0, 9 + 2

nd a novel 9 + 4 type with an apparent duplication of the cen-ral apparatus [32]. Recently, 9 + 2 and 9 + 4 axonemes were also

eported in mouse [33,34], suggesting that the simultaneous pres-nce of these three axoneme types may represent a common featuref LR cilia in mammals. Differences between mouse and rabbit thusostly relate to PNC morphology and to the size of the ciliated field

f cells (Fig. 2E).

l side up in (B). KV, GRP and PNC are highlighted in green. (E) Variation of size andn (medaka and rabbit numbers from [15], others from own analyses). n, notochord;0 �m (B + C) and 100 �m (E). (Panel A reprinted from [15] with permission from

1.2. Amphibians

Amphibians were considered to be the ‘thorn in the side of thecilia model’ by Tabin, the pioneer of molecular analysis of LR asym-metry, in a review in 2005 [13]. No flow had been described infrog embryos, however, early molecular and functional asymme-tries indicated symmetry breakage already at early cleavage stages[12]. Led by our comparative morphological analysis of mouseand rabbit, specifically the finding that the organizer was not theequivalent of the ciliated field of cells producing leftward flow inmammals, we reinvestigated X. laevis embryos focusing on the pos-terior notochord [27]. Cilia and dynein gene expression indeed arepresent at the posterior notochord of early neurula embryos, in astructure called gastrocoel roof plate (GRP), a ciliated epitheliumtransiently embedded into the dorsal endoderm of the archenteron(Figs. 1C and 2B [18,35]). Shook et al. described these cilia alreadyin 2004 and speculated about a functional role in LR axis determi-nation [36]. In dorsal explants we showed that cilia were motile,polarized to the posterior pole and rotated in a clockwise mannerto produce a leftward fluid flow across the GRP [18]. The func-tional relevance of leftward flow in X. laevis was demonstrated byexperiments in which flow was inhibited by injections of a highlyviscous methylcellulose solution into the archenteron, a manipula-tion which resulted in absence of marker gene expression and organsitus [18]. Knockdown experiments, in which morpholino antisenseoligonucleotides for number of conserved ciliary LR genes weretargeted to the GRP, in all cases affected flow and consequently tad-pole laterality (our unpublished results). These results demonstratethat the GRP-cilia-flow module is homologous to the PNC-cilia-flowmodule of the mammals.

We have in the meantime extended our analysis to two furtheramphibian species, another anuran, the diploid frog X. tropicalis, and

the urodele Ambystoma mexicanum, the axolotl. Despite large dif-ferences in size, the overall architecture of the early neurula embryois quite similar to X. laevis (Figs. 1C–E and 2E). In both cases a GRPwas present (Fig. 1D and E), cells were ciliated (insets in Fig. 1D andE) and produced a leftward flow (not shown).

M. Blum et al. / Seminars in Cell & Developmental Biology 20 (2009) 464–471 467

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ig. 3. The laterality coordinator. Schematic representation of an idealized vertebraty Nodal expressing cells (blue) on either side, and situated in the roof of the archensuing tilt and the clockwise rotation of cilia result in a net leftward flow of extrac

.3. Fish

.3.1. Teleost fishLeftward flow was reported in two teleost species, zebrafish

16,17] and medaka [15]. The ciliated epithelium in these casesocalizes to Kupffer’s vesicle (KV), an enigmatic and transienttructure which first appears at the end of gastrulation in theailbud, posterior to the notochord (Figs. 1F, F′ and 2A). KV mor-hologies differ slightly between zebrafish and medaka. While

n zebrafish the KV is spherical and cilia and flow are found allround the sphere [37], the medaka KV appears dome-shapednd motile cilia were found on the concave surface [15]. Like inammals and amphibians, leftward flow represents the symmetry

reaking event. Mutants lacking a KV (no tail-Brachyury; [38]) orisplaying a small and malformed KV (floating head-noto; [38]) areharacterized by randomization of organ situs. Gene knockdownxperiments, which in zebrafish can be readily targeted to the KVecause of the high endocytotic activity of KV precursor cells [39],emonstrated that the same set of conserved LR genes as in mousend Xenopus rule ciliogenesis and flow in zebrafish [16,40,41].

.3.2. Chondrostean fishZebrafish and medaka belong to two highly derived groups of

eleostean fish (Cypriniformes, carps, and Beloniformes, needlefish,espectively). In order to verify the role of leftward flow for all ray-nned fishes, analysis of more basal taxa would be desirable. Flowas unfortunately not analyzed in any basal fish species so far. An

n-depth description of gastrulation and mesoderm morphogene-is in the white sturgeon (Acipenser transmontanus), a member ofhe most basal group of ray-finned fish, the Chondrostei, has how-ver been published by Jessica Bolker back in 1993 [42]. In contrast

o teleosteans, which cleave superficially with the embryo sittingn a large single yolk cell, chondrosteans show holoblastic cleav-ge and also a gastrulation mode similar to amphibians. They werehus considered an excellent taxonomic outgroup for the amphib-ans [43–45]. Strikingly, sturgeon embryos possess a bona fide GRP

rality coordinator. A field of cells with posteriorly polarized cilia (green) is bordered. Ventral view from posterior (front) to anterior (back). Posterior polarization, the

r fluid (arrow). Inset highlights tilt and rotation of a single ciliated cell.

([42]; Fig. 1G and G′). Close inspection of the original scanningelectron microscopic pictures clearly reveal the presence of ciliaon GRP cells, although these photographs were not focused ontocilia (inset in Fig. 1G). It seems a safe bet to predict that a leftwardflow should be present at the GRP of the white sturgeon embryosas well. Short of this final proof we still like to suggest that theteleost KV evolved from the GRP of a common ancestor of ray-finned fishes. Thus, KV, GRP and PNC should represent homologousembryonic structures which function in LR axis determination ofall vertebrates.

1.4. Common denominator of vertebrate flow

Comparison of flow in mammals, amphibians and fish revealsa number of common features. An idealized ‘laterality coordinator’(for terminology see Section 1.5 below) is schematically depicted inFig. 3. PNC/GRP/KV all represent transient structures, which format the posterior end of the notochord in late gastrula/early neurulaembryos and disappear before the onset of organogenesis. Mostcells end up in the notochord; in frog and fish contributions to thesomites have been described as well [36,46]. There is no single genewhich unequivocally marks PNC/GRP/KV. However, Nodal acts as atell-tale gene to locate the ciliated epithelia in all cases [27,47,48](Fig. 3). Expression borders – and thus highlights – PNC/GRP/KV onthe left and right side in a bilateral pattern (Nodal in mammals, Xnr1in X. laevis and spaw in zebrafish). PNC/GRP/KV represent fields ofmonociliated cells of varying size and shape (Fig. 2E; Fig. 3). Ciliagrow to comparable length, with an average of 5 �m. Shortly aftertheir first appearance, cilia polarize to the posterior pole of cells(Fig. 3). The resulting tilt, together with their clockwise rotationalbeat pattern, leads to the observed robust flow of extracellular fluid

from right to left (Fig. 3), which in all cases precedes the onset ofasymmetric Nodal transcription in the left lateral plate mesodermby a few hours [30,31].

In addition to these more descriptive parameters, three verysignificant common embryological characteristics apply to PNC,

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68 M. Blum et al. / Seminars in Cell & D

RP and KV. (1) Although of mesodermal origin and fate, PNC,RP and KV are derived from surface epithelial cells. The PNC

s derived from the epiblast [36]. During gastrulation the PNC ishe third tissue to leave the node anteriorly, following the pre-hordal and notochordal cells [18,24]. The amphibian and sturgeonRP is derived from the superficial mesoderm, i.e. a tissue whichriginates from the outer epithelial layer of the blastula and invagi-ates over the dorsal lip of the blastopore following the prechordalesoderm and the anterior notochord [36,42]. The teleost KV is

erived from the so-called dorsal forerunner cells (DFCs), meso-ermal cells which epithelialize to give rise to the KV [40,46]. Aecent live multi-photon confocal imaging study has revealed thatFCs in turn are derived from dorsal surface epithelial cells, which

ngress to give rise to DFCs [49]. Thus, PNC/GRP/KV cells all shoulde derived from superficial mesoderm. (2) Though clearly differ-

ng in size and shape, PNC, GRP and KV in fact all represent partsr remnants of the gastrocoel/archenteron. The PNC in mouse, i.e.he distal indentation most commonly referred to as “node”, haseen called “archenteron” until 1991, for example in Theiler’s atlasf the house mouse [50]. The name unfavorably has been aban-oned in a search for a murine equivalent of the amphibian dorsal

ip and the chick Hensen’s node ([51]; see also our detailed dis-ussion of this issue in [27]). During teleostean fish evolution,mbryonic cavities such as blastocoel and archenteron were largelyliminated in the course of a compaction of embryonic germ lay-rs into new arrangements ([45] and references therein). The KVas long been considered to represent the evolutionary remnantf the posterior archenteron and neuroenteric canal ([45,52] andeferences therein). (3) The sole function of the retention of super-cial mesoderm in the epithelial surface of the gastrocoel roof,

.e. of PNC/GRP/KV, an obviously ancestral vertebrate character, ishe breakage of the bilateral symmetry in the vertebrate gastrulambryo. This proposal has so far been experimentally tested inebrafish embryos by laser ablation [16] and in Medaka by man-al disruption of the KV [53]. Preliminary experiments in X. laevisuggest that manual ablation of superficial mesoderm exclusivelyffects LR development (our unpublished results). PNC ablationxperiments in mouse have been reported by Tam and coworkers, inhich exclusively laterality and somitogenesis were affected [54].

n summary, leftward flow is the decisive step in vertebrate symme-ry breakage and occurs in a ciliated field of cells, which is localizedn the gastrocoel/archenteron and derived from superficial meso-erm.

.5. Terminology

Presently, most authors use the terms ‘node’/‘ventral node’ ornode equivalent’, ‘nodal flow’ and ‘nodal cilia’ to address struc-ure, event and the specifics of the involved cilia. In the light of thebove reasoning, we consider this terminology confusing and inap-ropriate. We propose to use leftward flow instead of ‘nodal flow’,nd Left-Right cilia instead of ‘nodal cilia’. The term ‘node’, we sug-est, should also be abandoned, in particular because in our mindt should be reserved to the denomination of the primary embry-nic organizer of the mammals (Spemann’s organizer). Althoughn all cases the ciliated field is represented by an archenteron rooflate, we propose to use PNC in mammals, GRP in amphibians andasal fish with amphibian-type gastrulation like sturgeons, and KVor teleosteans. In order to come up with a term applicable to allpecies ‘laterality organ’ or ‘left-right organizer’ have been pre-iously suggested by others. We do not consider PNC/GRP/KV as

rgans, as they exist only very transiently and consist of just oneissue. An organizer function has not been described, for exampley transplantation experiments of the KV. As an alternative term weike to propose ‘laterality coordinator’, as this term encompasses allspects of the process.

mental Biology 20 (2009) 464–471

2. The chick problem

Molecular analysis of LR axis formation has been initiated in thechick embryo by Tabin and coworkers in 1995, when the first asym-metrically expressed genes were detected [55]. Despite the powerof mouse and zebrafish genetics, due to the manipulative poten-tial of the chick it still is one of the best studied model organismsin the LR field. Dynein gene expression and tubulin immunohisto-chemistry have indicated a possible role for cilia at gastrula stages[35]. What the chick still lacks, however, are LR cilia and leftwardflow. Scanning electron microscopic studies from stage 4 (node for-mation) to stage 7 (first pair of somites), i.e. directly before andduring induction of the Nodal cascade, failed to detect any can-didate ciliated cells ([56] and our own unpublished results). Inaddition, the tell-tale marker gene Nodal shows left-sided expres-sion at Hensen’s node [55] and only becomes bilaterally activatedonce the Nodal cascade is fully active in the left lateral plate meso-derm at stage 9. Indicative genes involved in ciliogenesis or LR ciliafunction are mostly not expressed during the relevant stages (notshown).

What the chick has instead is a unilateral left-sided midlineNodal domain [55] and a morphologically asymmetrical Hensen’snode [57,58]. As a consequence of this node asymmetry, there aregenes expressed in the primitive streak which display right asym-metries at the node (such as FGF8; [59]), and genes expressed inthe notochord and floor plate with respective left asymmetries (i.e.Shh or Foxa2 [55,60]). Together, these molecular asymmetries at themidline are sufficient to induce the Nodal cascade in the left LPMin a robust manner. Therefore, node asymmetry in the chick mani-fests symmetry breakage without involvement of cilia and flow. Themolecular and cellular mechanisms underlying the morphogeneticprocess which renders the node asymmetrical, however, have notbeen elucidated as yet. We like to speculate that signaling path-ways and cellular mechanisms involved in setting up the lateralitycoordinator and flow in other vertebrates might be involved in thisprocess as well, and that flow was lost secondarily once the nodebecame asymmetrical in the course of chick evolution.

One striking feature of the chick, which might explain theabsence of flow, is its apparent lack of a superficial mesoderm, asrecently pointed out by Keller and coworkers ([36]; see also [61,62]).Other birds have not been analyzed so far, which is why Keller liststhem as ambiguous cases [36]. It might be worthwhile to look atprimitive birds, for example flightless ones like ostriches [63]. Onthe other hand, as birds on the whole represent a highly derivedgroup of sauropsids, basal taxa such as crocodiles and turtles mightbe promising candidates, in particular as they possess superficialmesoderm [36]. Such an analysis would tell when flow was evo-lutionary lost during bird evolution, at the base of bird evolutionor already in the stem reptiles after the mammals had branchedoff.

Taken together, absence of flow in chick does not present a caseto question leftward flow as an ancestral mode of symmetry break-age in the vertebrates. The presence of GRP and cilia in the whitesturgeon and in amphibians, as well as in teleosteans and mam-mals clearly demonstrates that flow was present at the base of thevertebrates already.

3. Primitive chordates: predictions for amphioxus

An asymmetric Nodal cascade was described in the tuni-cates Ciona intestinalis, Botryllus schlosseri and Halocynthia

roretzi [8,64,65] and the cephalochordate Branchiostoma [8,9](amphioxus). Here we like to discuss amphioxus, as it is consid-ered to represent an ancestral state of chordate evolution, while themore derived tunicates have recently been grouped together withthe vertebrates [66,67]. As in vertebrates, Nodal is first expressed

M. Blum et al. / Seminars in Cell & Developmental Biology 20 (2009) 464–471 469

Fig. 4. Amphioxus and sea urchin. (A) Amphioxus. Schematic drawing of a neurula stage amphioxus embryo cut transversely (A) and mid-sagittally (A′). The bilateral tell-taleNodal expression indicated in blue (A) predicts an in-between GRP in the archenteron (green in A, A′). (B and C) Sea urchin. 3D schematic drawings of early (B and B′) and late(C and C′) gastrula sea urchin embryos in whole-mount (B and C) and bisected along the dorso-ventral (B′) and left-right (C′) axis, respectively. Proposed right half shown in(B′), and oral half shown in (C′), Early (dorsal/aboral) symmetric Nodal organizer domain in (B) and (B′) and late asymmetric expression domains in ectoderm and archenteroni iliateda ne exp[ ). a, ane axial

ittbpttwapthatTswa

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n (C) and (C′) are indicated in blue. The supposed position of a field of polarized cccount for (a) conserved left asymmetry of Nodal expression and (b) organizer ge72] (B and B′) and [10] (C and C′). Schemes were redrawn from [9] (A) and [73] (A′

ndoderm; l, left; n, notochord; nc, neuroenteric canal; ne, neuroectoderm; pm, par

n the amphioxus organizer (dorsal lip; [9,68]). During gastrula-ion, this domain invaginates and simultaneously splits, so that inhe 10 h neurula a left and right domain is observed, separatedy notochordal mesoderm [9]. The Nodal domain represents thearaxial mesoderm, derived from superficial cells like in the ver-ebrates (Fig. 4A and A′). This setting thus is quite reminiscent ofhe vertebrate laterality coordinator, i.e. a bilateral Nodal domainithin the archenteron, encasing notochordal mesoderm laterally

nd bordered by endodermal cells (Fig. 4A). Therefore, we like toredict that the notochordal cells harbor the laterality coordina-or and possess polarized cilia which produce a leftward flow. Ciliaave been described on the dorsal lip as well as on archenteral cellst late gastrula [69]. About 90 min after bilateral induction of Nodal,he right-sided domain diminishes and eventually disappears [9].his, we suggest, should be the result of leftward flow. The left-ided domain spreads in due course into all three germ layers asell as along the anterior–posterior axis, providing cues for later

symmetric morphogenesis.

. Deuterostomes: speculations about sea urchins

Echinoderms and hemichordates are considered to constitutehe monophyletic group of the Ambulacraria, based on molecularnd morphological criteria [11,66,67,70]. A Nodal cascade has beenescribed in sea urchins [3,10] and thus should also be present inemichordates. Nodal expression in sea urchins is seen in the oralctoderm of the blastula/gastrula [3,10]. At late gastrula, an asym-etric domain appears at the tip of the archenteron in a few cells,ostly (i.e. in about 70% of cases [10]) displaced to the right side

Fig. 4C and C′). Shortly thereafter, the ectodermal domain gets dis-laced to the right as well [10]. The archenteral tip cells should

e mesodermal in character, as they give rise to the coelomic sachich splits into the two pouches. If there was a flow, which we

ike to speculate, it should precede the asymmetric induction ofodal in the archenteron and be directed towards this domain.nother reason for this speculation, besides the mesodermal nature

cells is indicated in green. The conventional echinoderm body plan is inverted toression on the dorsal side. Expression domains are indicated according to [9] (A),terior; ar, archenteron; bc, blastocoel; bp, blastoporus; d, dorsal; ec, ectoderm; en,mesoderm; r, right; v, ventral.

of surface-derived archenteron tip cells, is the fact that monociliahave been found on archenteral cells in a crinoid, the feather starComanthus japonica [71].

The right-sided nature of Nodal expression might be considereda non-conserved aspect of LR asymmetry. If, however, asymmetricNodal expression was conserved and left-sided like in chordates (asdepicted in Fig. 4C and C′), the sole complication to the sea urchinbody plan would be that the mouth in the larva would open on thedorsal side instead of ventrally (Fig. 4). This possibility seems attrac-tive to us, as dorsal/organizer markers consistently are expressedin the oral ectoderm, i.e. Nodal, Antivin, Brachyury and Goosecoid[3,72]. Therefore, the archenteral cells on the oral side, derived fromoral/dorsal blastula cells, could be the ones becoming polarized andproducing a vectorial flow. Alternatively, if all archenteral cells wereciliated and polarized, these cells could be secreting a morphogen.This would correspond perfectly well with the situation in the ver-tebrates, where the superficial mesoderm cells, derived from thedorsal side, polarize posteriorly following their invagination ontothe dorsal side of the archenteron [36].

5. Conclusion/outlook

Cilia-driven leftward flow presents the symmetry breakingevent in the vertebrates (certainly), chordates (likely) and deuteros-tomes (maybe). During gastrulation, surface-derived mesodermalcells organize as a monociliated epithelium inside the archenteron.Cilia polarize towards the posterior pole of cells and due to theirrotational beat pattern produce a leftward flow in the extracel-lular space. Flow precedes asymmetric induction of the left-sidedNodal signaling cascade, which provides the cues for asymmetricmorphogenesis.

As new model organisms are being established, and moresophisticated techniques become available for live imaging of tinyembryos and fragments thereof, our proposals may soon becomeaccessible to experimental examination. Protostomes, albeit asym-metries have been described and new ones continue to be found,

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70 M. Blum et al. / Seminars in Cell & D

ikely do not use flow-based symmetry breakage. Though cilia arebundant, and planar cell polarity as the likely mechanism of ciliaolarization is probably widespread, no Nodal homolog has beenetected ever in a non-deuterostome embryo. Thus, evolution of theodal cascade and recruitment into a cilia-based signal transfer sys-

em should be the necessary steps for the appearance of flow-drivenR asymmetry at the base of the deuterostomes.

cknowledgements

We are grateful to Jessica Bolker for digging out and scanninger original SEM photographs of sturgeon GRPs, and to Nobutakairokawa for the kind supply of SEM pictures from medaka. TB andW like to thank Ray Keller and Kristen Kroll for their organiza-ion of the 2008 CSH Xenopus course, during which they analyzedenopus tropicalis and axolotl embryos. Kerstin Feistel provided theabbit and Philipp Andre the mouse SEMs, Bernd Schmid preparedll drawings. Axel Schweickert helped with the preparation of theanuscript, particularly Sections 3 and 4. Work in the Blum lab was

upported by grants from the Deutsche Forschungsgemeinschafto MB and Ph. D. fellowships from the Landesgraduiertenstiftungaden-Württemberg to PV, TW and TB.

eferences

[1] Schier AF. Nodal signaling in vertebrate development. Annu Rev Cell Dev Biol2003;19:589–621.

[2] Hirokawa N, Tanaka Y, Okada Y, Takeda S. Nodal flow and the generation ofleft-right asymmetry. Cell 2006;125(1):33–45.

[3] Duboc V, Lepage T. A conserved role for the Nodal signaling pathway in theestablishment of dorso-ventral and left-right axes in deuterostomes. J ExpZoolog B Mol Dev Evol 2008;310(1):41–53.

[4] Boorman CJ, Shimeld SM. The evolution of left-right asymmetry in chordates.Bioessays 2002;24(11):1004–11.

[5] Chea HK, Wright CV, Swalla BJ. Nodal signaling and the evolution of deuteros-tome gastrulation. Dev Dyn 2005;234(2):269–78.

[6] Aizawa H, Goto M, Sato T, Okamoto H. Temporally regulated asymmetric neuro-genesis causes left-right difference in the zebrafish habenular structures. DevCell 2007;12(1):87–98.

[7] Kuan YS, Yu HH, Moens CB, Halpern ME. Neuropilin asymmetry mediates a left-right difference in habenular connectivity. Development 2007;134(5):857–65.

[8] Boorman CJ, Shimeld SM. Pitx homeobox genes in Ciona and amphioxus showleft-right asymmetry is a conserved chordate character and define the ascidianadenohypophysis. Evol Dev 2002;4(5):354–65.

[9] Yu JK, Holland LZ, Holland ND. An amphioxus nodal gene (AmphiNodal)with early symmetrical expression in the organizer and mesoderm and laterasymmetrical expression associated with left-right axis formation. Evol Dev2002;4(6):418–25.

10] Duboc V, Rottinger E, Lapraz F, Besnardeau L, Lepage T. Left-right asymmetryin the sea urchin embryo is regulated by Nodal signaling on the right side. DevCell 2005;9(1):147–58.

11] Swalla BJ. Building divergent body plans with similar genetic pathways. Hered-ity 2006;97(3):235–43.

12] Levin M. Left-right asymmetry in embryonic development: a comprehensivereview. Mech Dev 2005;122(1):3–25.

13] Tabin C. Do we know anything about how left-right asymmetry is first estab-lished in the vertebrate embryo? J Mol Histol 2005;36(5):317–23.

14] Nonaka S, Tanaka Y, Okada Y, Takeda S, Harada A, Kanai Y, et al. Ran-domization of left-right asymmetry due to loss of nodal cilia generatingleftward flow of extraembryonic fluid in mice lacking KIF3B motor protein. Cell1998;95(6):829–37.

15] Okada Y, Takeda S, Tanaka Y, Belmonte JC, Hirokawa N. Mechanism of nodalflow: a conserved symmetry breaking event in left-right axis determination.Cell 2005;121(4):633–44.

16] Essner JJ, Amack JD, Nyholm MK, Harris EB, Yost HJ. Kupffer’s vesicle is a ciliatedorgan of asymmetry in the zebrafish embryo that initiates left-right develop-ment of the brain, heart and gut. Development 2005;132(6):1247–60.

17] Kramer-Zucker AG, Olale F, Haycraft CJ, Yoder BK, Schier AF, Drummond IA.Cilia-driven fluid flow in the zebrafish pronephros, brain and Kupffer’s vesicleis required for normal organogenesis. Development 2005;132(8):1907–21.

18] Schweickert A, Weber T, Beyer T, Vick P, Bogusch S, Feistel K, et al. Cilia-drivenleftward flow determines laterality in Xenopus. Curr Biol 2007;17(1):60–6.

19] Kramer KL, Barnette JE, Yost HJ. PKCgamma regulates syndecan-2 inside-out

signaling during Xenopus left-right development. Cell 2002;111(7):981–90.

20] Fukumoto T, Kema IP, Levin M. Serotonin signaling is a very early stepin patterning of the left-right axis in chick and frog embryos. Curr Biol2005;15(9):794–803.

21] Levin M, Mercola M. Gap junctions are involved in the early generation of left-right asymmetry. Dev Biol 1998;203(1):90–105.

[

[

mental Biology 20 (2009) 464–471

22] Levin M, Thorlin T, Robinson KR, Nogi T, Mercola M. Asymmetries in H+/K+-ATPase and cell membrane potentials comprise a very early step in left-rightpatterning. Cell 2002;111(1):77–89.

23] Afzelius BA. A human syndrome caused by immotile cilia. Science 1976;193(4250):317–9.

24] Sulik K, Dehart DB, Iangaki T, Carson JL, Vrablic T, Gesteland K, et al. Morphogen-esis of the murine node and notochordal plate. Dev Dyn 1994;201(3):260–78.

25] Raya A, Belmonte JC. Left-right asymmetry in the vertebrate embryo: from earlyinformation to higher-level integration. Nat Rev Genet 2006;7(4):283–93.

26] Nonaka S, Shiratori H, Saijoh Y, Hamada H. Determination of left-right pattern-ing of the mouse embryo by artificial nodal flow. Nature 2002;418(6893):96–9.

27] Blum M, Andre P, Muders K, Schweickert A, Fischer A, Bitzer E, et al. Ciliationand gene expression distinguish between node and posterior notochord in themammalian embryo. Differentiation 2007;75(2):133–46.

28] Hensen V. Beobachtungen ueber die Befruchtung und Entwicklung des Kan-inchens and Meerschweinchens. Z Anat Entwicklungsgesch 1876;1. p. 213–273,353–423.

29] Viebahn C, Mayer B, Miething A. Morphology of incipient mesoderm formationin the rabbit embryo: a light- and retrospective electron-microscopic study.Acta Anat (Basel) 1995;154(2):99–110.

30] Cartwright JH, Piro O, Tuval I. Fluid-dynamical basis of the embryonic devel-opment of left-right asymmetry in vertebrates. Proc Natl Acad Sci USA2004;101(19):7234–9.

31] Nonaka S, Yoshiba S, Watanabe D, Ikeuchi S, Goto T, Marshall WF, et al. Denovo formation of left-right asymmetry by posterior tilt of nodal cilia. PLoS Biol2005;3(8):e268.

32] Feistel K, Blum M. Three types of cilia including a novel 9 + 4 axoneme on thenotochordal plate of the rabbit embryo. Dev Dyn 2006;235(12):3348–58.

33] Caspary T, Larkins CE, Anderson KV. The graded response to Sonic Hedgehogdepends on cilia architecture. Dev Cell 2007;12(5):767–78.

34] Lee JD, Anderson KV. Morphogenesis of the node and notochord: the cellularbasis for the establishment and maintenance of left-right asymmetry in themouse. Dev Dyn 2008;237(12):3464–76.

35] Essner JJ, Vogan KJ, Wagner MK, Tabin CJ, Yost HJ, Brueckner M. Conservedfunction for embryonic nodal cilia. Nature 2002;418(6893):37–8.

36] Shook DR, Majer C, Keller R. Pattern and morphogenesis of presumptive super-ficial mesoderm in two closely related species, Xenopus laevis and Xenopustropicalis. Dev Biol 2004;270(1):163–85.

37] Kreiling JA, Williams G, Creton R. Analysis of Kupffer’s vesicle in zebrafishembryos using a cave automated virtual environment. Dev Dyn 2007;236(7):1963–9.

38] Melby AE, Warga RM, Kimmel CB. Specification of cell fates at the dorsal marginof the zebrafish gastrula. Development 1996;122(7):2225–37.

39] Amack JD, Yost HJ. The T box transcription factor no tail in ciliated cells controlszebrafish left-right asymmetry. Curr Biol 2004;14(8):685–90.

40] Amack JD, Wang X, Yost HJ. Two T-box genes play independent and cooperativeroles to regulate morphogenesis of ciliated Kupffer’s vesicle in zebrafish. DevBiol 2007;310(2):196–210.

41] Bisgrove BW, Snarr BS, Emrazian A, Yost HJ. Polaris and Polycystin-2 in dor-sal forerunner cells and Kupffer’s vesicle are required for specification of thezebrafish left-right axis. Dev Biol 2005;287(2):274–88.

42] Bolker JA. Gastrulation and mesoderm morphogenesis in the white sturgeon. JExp Zool 1993;266(2):116–31.

43] Bolker JA. Comparison of gastrulation in frogs and fish. Am Zool 1994;34:313–22.

44] Collazo A, Bolker J, Keller R. A phylogenetic perspective on teleost gastrulation.Am Nat 1994;144:133–52.

45] Cooper MS, Virta VC. Evolution of gastrulation in the ray-finned (actinoptery-gian) fishes. J Exp Zoolog B Mol Dev Evol 2007;308(5):591–608.

46] Cooper MS, D’Amico LA. A cluster of noninvoluting endocytic cells at the marginof the zebrafish blastoderm marks the site of embryonic shield formation. DevBiol 1996;180(1):184–98.

47] Long S, Ahmad N, Rebagliati M. The zebrafish nodal-related gene southpawis required for visceral and diencephalic left-right asymmetry. Development2003;130(11):2303–16.

48] Lowe LA, Supp DM, Sampath K, Yokoyama T, Wright CV, Potter SS, et al. Con-served left-right asymmetry of nodal expression and alterations in murine situsinversus. Nature 1996;381(6578):158–61.

49] Oteiza P, Koppen M, Concha ML, Heisenberg CP. Origin and shaping of the lat-erality organ in zebrafish. Development 2008;135(16):2807–13.

50] Theiler K. The house mouse. Berlin/Heidelberg/New York: Springer; 1972.51] Beddington RS, Jaenisch R, Smith AB, McLaren AL, Lawson KA, Herrmann BG.

Three-dimensional representation of gastrulation in the mouse. Ciba FoundSymp 1992;165:55–60.

52] D’Amico LA, Cooper MS. Spatially distinct domains of cell behavior in thezebrafish organizer region. Biochem Cell Biol 1997;75(5):563–77.

53] Bajoghli B, Aghaallaei N, Soroldoni D, Czerny T. The roles of Groucho/Tlein left-right asymmetry and Kupffer’s vesicle organogenesis. Dev Biol2007;303(1):347–61.

54] Davidson BP, Kinder SJ, Steiner K, Schoenwolf GC, Tam PP. Impact of node abla-

tion on the morphogenesis of the body axis and the lateral asymmetry of themouse embryo during early organogenesis. Dev Biol 1999;211(1):11–26.

55] Levin M, Johnson RL, Stern CD, Kuehn M, Tabin C. A molecular pathway deter-mining left-right asymmetry in chick embryogenesis. Cell 1995;82(5):803–14.

56] Manner J. Does an equivalent of the “ventral node” exist in chick embryos? Ascanning electron microscopic study. Anat Embryol (Berl) 2001;203(6):481–90.

evelop

[[

[

[

[

[

[

[

[

[

[

[

[

[

[of Comanthus japonica (Echinodermata: Crinoidea). Tissue Cell 1976;8(3):

M. Blum et al. / Seminars in Cell & D

57] Cooke J. Vertebrate embryo handedness. Nature 1995;374(6524):681.58] Dathe V, Gamel A, Manner J, Brand-Saberi B, Christ B. Morphological left-right

asymmetry of Hensen’s node precedes the asymmetric expression of Shh andFgf8 in the chick embryo. Anat Embryol (Berl) 2002;205(5–6):343–54.

59] Boettger T, Wittler L, Kessel M. FGF8 functions in the specification of the rightbody side of the chick. Curr Biol 1999;9(5):277–80.

60] Levin M. The roles of activin and follistatin signaling in chick gastrulation. Int JDev Biol 1998;42(4):553–9.

61] Jurand A. The development of the notochord in chick embryos. J Embryol ExpMorphol 1962;10:602–21.

62] Sausedo RA, Schoenwolf GC. Cell behaviors underlying notochord formationand extension in avian embryos: quantitative and immunocytochemical stud-ies. Anat Rec 1993;237(1):58–70.

63] Hackett SJ, Kimball RT, Reddy S, Bowie RC, Braun EL, Braun MJ, et al.A phylogenomic study of birds reveals their evolutionary history. Science2008;320(5884):1763–8.

64] Morokuma J, Ueno M, Kawanishi H, Saiga H, Nishida H. HrNodal, the ascid-ian nodal-related gene, is expressed in the left side of the epidermis, and liesupstream of HrPitx. Dev Genes Evol 2002;212(9):439–46.

65] Tiozzo S, Christiaen L, Deyts C, Manni L, Joly JS, Burighel P. Embryonic ver-sus blastogenetic development in the compound ascidian Botryllus schlosseri:insights from Pitx expression patterns. Dev Dyn 2005;232(2):468–78.

[

[

mental Biology 20 (2009) 464–471 471

66] Delsuc F, Brinkmann H, Chourrout D, Philippe H. Tunicates and not cephalo-chordates are the closest living relatives of vertebrates. Nature 2006;439(7079):965–8.

67] Bourlat SJ, Juliusdottir T, Lowe CJ, Freeman R, Aronowicz J, Kirschner M, et al.Deuterostome phylogeny reveals monophyletic chordates and the new phylumXenoturbellida. Nature 2006;444(7115):85–8.

68] Yu JK, Satou Y, Holland ND, Shin IT, Kohara Y, Satoh N, et al. Axial patterning incephalochordates and the evolution of the organizer. Nature 2007;445(7128):613–7.

69] Hirakow R, Kajita N. Electron microscopic study of the development ofamphioxus, Branchiostoma belcheri tsingtauense: the gastrula. J Morphol 1991;207:37–52.

70] Smith AB. Deuterostomes in a twist: the origins of a radical new body plan. EvolDev 2008;10(4):493–503.

71] Holland ND. The fine structure of the embryo during the gastrula stage

491–510.72] Duboc V, Rottinger E, Besnardeau L, Lepage T. Nodal and BMP2/4 signaling orga-

nizes the oral-aboral axis of the sea urchin embryo. Dev Cell 2004;6(3):397–410.

73] Conklin EG. The embryology of amphioxus. J Morph 1932;54(1):69–151.