developmental mechanism and evolutionary origin of vertebrate left/right asymmetries

Developmental mechanism and evolutionary

origin of vertebrate left/right asymmetries

Jonathan Cooke*

Department of Zoology and Museum of Comparative Zoology, University of Cambridge, Downing Street, Cambridge, UK

(E-mail : [email protected])

(Received 18 October 2002; revised 27 May 2003; accepted 28 May 2003)

ABSTRACT

The systematically ‘handed’, or directionally asymmetrical way in which the major viscera are packed within thevertebrate body is known as situs. Other less obvious vertebrate lateralisations concern cognitive neural function,and include the human phenomena of hand-use preference and language-associated cognitive partitioning. Anoverview, rather than an exhaustive scholarly review, is given of recent advances in molecular understanding ofthe mechanism that ensures normal development of ‘correct ’ situs. While the asymmetry itself and its left/rightdirection are clearly vertebrate-conserved characters, data available from various embryo types are comparedin order to assess the likelihood that the developmental mechanism is evolutionarily conserved in its entirety.A conserved post-gastrular ‘phylotypic ’ stage, with left- and right-specific cascades of key, orthologous geneexpressions, clearly exists. It now seems probable that earlier steps, in which symmetry-breaking information isreliably transduced to trigger these cascades on the correct sides, are also conserved at depth although it remainsunclear exactly how these steps operate. Earlier data indicated that the initiation of symmetry-breaking had beentransformed, among the different vertebrate classes, as drastically as has the anatomy of pre-gastrular develop-ment itself, but it now seems more likely that this apparent diversity is deceptive.

Ideas concerning the functional advantages to the vertebrate lifestyle of a systematically asymmetrical visceralpacking arrangement, while untestable, are accepted because they form a plausible adaptationist ‘ just-so ’ story.Nevertheless, two contrasting beliefs are possible about the evolutionary origins of situs. Major recent advances inanalysis of its developmental mechanism are largely due not to zoologists, comparative anatomists or evolution-ary systematists, but to molecular geneticists, and these workers have generally assumed that the asymmetry is anevolutionary novelty imposed on a true bilateral symmetry, at or close to the origin of the vertebrate clade. Amajor purpose of this review is to advocate an alternative view, on the grounds of comparative anatomy andmolecular systematics together with the comparative study of expressions of orthologous genes in different forms.This view is that situs represents a co-optation of a pre-existing, evolutionarily ancient non-bilaterality of the adultform in a vertebrate ancestor. Viewed this way, vertebrate or chordate origins are best understood as the novelimposition of an adaptively bilateral locomotory-skeletal-neural system, around a retained non-symmetrical‘visceral ’ animal.

One component of neuro-anatomical asymmetry, the habenular/parapineal one that originates in the dien-cephalon, has recently been found (in teleosts) to be initiated from the same ‘phylotypic ’ gene cascade thatcontrols situs development. But the function of this particular diencephalic asymmetry is currently unclear. Otherleft-right partitionings of brain function, including the much more recently evolved, cerebral cortically locatedone associated with human language and hand-use, may be controlled entirely separately from situs even thoughtheir directionality has a particular relation to it in a majority of individuals.

Finally, possible relationships are discussed between the vertebrate directional asymmetries and those thatoccur sporadically among protostome bilaterian forms. These may have very different evolutionary and mol-ecular bases, such that there may have been constraints, in protostome evolution, upon any exploitation of leftand right for complex organismic, and particularly cognitive neural function.

Key words : handedness, left-right asymmetry, evolution, development, phylogeny.

* Address for correspondence : 10, Danvers Road, London N8 7HH, UK.

Biol. Rev. (2004), 79, pp. 377–407. f Cambridge Philosophical Society 377DOI: 10.1017/S1464793103006298 Printed in the United Kingdom

CONTENTS

I. Introduction: Scope and level of this review ........................................................................................... 378II. Overview of the developmental mechanism for visceral situs ................................................................ 379

(1) A vertebrate-conserved ‘phylotypic ’ post-gastrular sector .............................................................. 379(2) Initial left-right symmetry breaking in earlier development : apparent diversification in

timing and in upstream cascade steps ................................................................................................ 382(3) Is there a unitary mechanism for developmental origin of vertebrate situs? ................................ 387(4) Changing left-right co-options of gene orthologues during vertebrate diversification, and

other puzzles .......................................................................................................................................... 388III. Viewpoints on the evolutionary origins of situs ....................................................................................... 389

(1) The functional ‘ just-so’ story: situs as a vertebrate (chordate) invention ...................................... 389(2) A constrasting view: situs as the co-optation of a pre-existing axis ................................................ 391

IV. Visceral major organ, neuro-anatomical and neuro-functional left and right : unitary orindependent origins? ................................................................................................................................... 395(1) Hominoid hand-use/linguistic lateralisation: origin and inheritance ........................................... 395(2) Hand-use/linguistic lateralisation and situs in development ........................................................... 397(3) Status of other vertebrate functional forebrain lateralisations ........................................................ 398

V. Implications for directional asymmetries in protostome animals ......................................................... 399VI. Conclusions .................................................................................................................................................. 400VII. Acknowledgements ...................................................................................................................................... 402VIII. References .................................................................................................................................................... 402

I. INTRODUCTION: SCOPE AND LEVEL OF

THIS REVIEW

Despite the nearly perfect bilateral symmetry of vertebrateaxial anatomy, paired sense organs and limbs, it is widelyrecognised even by non-biologists that the major visceralorgans are packed into the human body cavity in a highlyasymmetrical manner, and that this is a ‘handed’ asym-metry in that it follows the same left-right direction(technically known as normal situs or situs solitus) in the vastmajority of individuals. The rare human situs inversushowever, the complete mirror-reversal of normal visceralanatomy, is a perfectly viable state of affairs (Torgerson,1950), though the decidedly less rare heterotaxias, confusionsof left-right anatomy within individuals, are usually patho-genic. It becomes clear, from comparative study of a rangeof vertebrate embryos, that while the developed viscera andparticularly the heart and great blood vessels have becomemore complex in function and anatomy, the directionality ofthe situs asymmetry is vertebrate-conserved.

Such directional asymmetry is quite distinct from thephenomena of fluctuating asymmetry and antisymmetry. Inthe former, limitations of ‘buffering’ of the developmentalmechanism against (genetically or environmentally con-ditioned) perturbation limit the precision with which rightand left ‘ symmetrical ’ structures can be mirror replicaswithin individual bodies. In antisymmetry, competitivesuppressive mechanisms ensure that right and left structuresare very different in size etc. (as in certain arthropodappendage pairs), but with essentially random ‘handedness ’.Certain more subtle directional asymmetries within ver-tebrate structure are reliably co-ordinated with, and couldthus be considered part of, situs. The preferred direction ofaxial torsion within the uterus or egg-shell, seen in mosttypes of vertebrate embryo, is perhaps the clearest example,but others may be the antero-posterior staggering or

alternation of the primitive left and right somite boundariesthat is clearest in acraniates (Wada, Garcia-Fernandez &Holland, 1999) but detectable in vertebrates ( J. Cooke,unpublished observatons on chick and frog embryos), andleft-right differences in both developmental and adultbranchial anatomy of some agnathan vertebrates.

Less universal but still highly significant is the preponder-ance (around 9:1) of right-hand-use preference for humanskilled activites ; the most conventional use of the term‘handedness ’. This is strongly (but not universally) linkedwith the phenomenon that most of us have the capacity forlanguage use preferentially located in our left cerebralcortices, with other, harder-to-define but complementarycognitive functions located on the right (e.g. McManus,1991). Thus it is only mildly incorrect to speak, in humans,of a particular ‘normal ’ relationship between visceral situs,hand-use preference and differential placement of highercognitive capacities within the hemispheres, although in factvarious ‘discordancies ’ among these are common and notstrongly dysfunctional.

Left-right functional complementarities of brain usage,with a preponderant directionality corresponding to thehuman one, have recently been found to characterise mostof the hominoid (human-ape) clade, and are probablyrelated to the onset of language-like cognitive abilities andassociated differential hand use (Annett & Annett, 1991;Cantalupo & Hopkins, 2001). But in addition to this, there isa whole range of phenomena involving differential function-ing, sometimes linked to detectably different anatomy, ofthe two sides of the brain in fish, reptiles, birds and non-primate mammals (see Section IV and references). For mostof these, however, it is unclear whether population levels ofdirectionality are similar to those of human hand preferenceetc., or show the much higher reliability characterising situs(see later discussion). Well-known adaptive idiosyncracies,such as the asymmetrical aural apparatus of owls, are very

378 Jonathan Cooke

probably extensions of, and thus capitalisations on, situs itselfalthough this is not positively known.

Molecular understanding of developmental mechanismshas recently made explosive advances for situs itself, andthese mechanisms, including data from all the experimental‘model ’ vertebrate embryo types, have been comprehen-sively reviewed for developmental specialists by severalauthors (cited in Section II.1). The aim of the present articleis not to replicate or even update these reviews but instead tosurvey the field for a broader readership, and in relation tothree key questions of evolutionary interest ; the degree ofconservation or universality of the whole situs developmentalcascade, the evolutionary origin of situs, and finally thedegrees of developmental and thus evolutionary relationshipbetween situs and the various neural lateralisations.

There is clear, vertebrate-wide conservation of a post-gastrular, ‘phylotypic ’ left-right organisation of the embryo,with distinctive left-hand and right-hand expressions of a setof gene orthologues. These initiate the differentials ingrowth and morphogenetic movement that characterisevisceral organ primordia on either side of the midline toproduce situs anatomy. But dramatic apparent differences inthe events that lead up to this stage, among vertebrateembryo types, have led to suggestions that the earlier sectorof mechanism, that which breaks the embryo’s apparentbilateral symmetry in the first place, has varied opportun-istically as much as has pre-gastrulation anatomy duringvertebrate evolution. While acknowledging that this mayturn out to be the case, I nevertheless propose that currentdata are consistent with in-depth conservation of even thisearly, so-called symmetry-breaking sector of mechanism.

Recently, experimental gene expression data in zebrafish(Danio rerio) have linked the origin of an anatomical brainasymmetry, the diencephalon-associated habenular andparapineal one that is probably of functional importance infish, amphibians, reptiles and birds, with the phylotypic situsgene cascade itself (Concha et al., 2000; Liang et al., 2000).However, a wider variety of documented left-right par-titionings of vertebrate forebrain function are not necessarilyall linked with this particular feature ; they remain to befitted into what traditional developmental geneticists wouldhave referred to as a ‘pedigree of causes ’ underlyingvertebrate asymmetries. Current data seem to link togetherhuman hand-use preference and higher cognitive left-rightpartitioning, as being controlled probabilistically by thesame system of left-right developmental information (seeSection IV and references). But strikingly, the data alsoindicate that this particular neural lateralisation system isquite independent from that controlling situs (Torgerson,1950; Kennedy et al., 1999; Tanaka et al., 1999).

Evolutionarily speaking, ‘explanation’ for a consistentlylateralised arrangement of vertebrate visceral packing is nothard to find. Given the material possibility, evolution can beexpected to have simulated optimal engineering design, andthe argument that such an arrangement is indeed optimalfor the vertebrate will be laid out in Section III. But, withthe interesting exception of at least some bird gastrulae(see Section III.2), extant vertebrates appear bilaterally sym-metrical in cellular structure as early embryos. It has thusbeen easy to make the assumption that the mechanism for

breaking this symmetry with a reliable left-right direction-ality was a novel ‘ invention’, selected for by the distinctivelifestyle requirements of an organism at or relatively close tothe origins of the vertebrate clade.

The present article re-emphasises data from zoology,comparative anatomy and comparative gene expressionstudies that advocate a different evolutionary scenario.These data suggest that a vertebrate ancestor possessed anevolutionarily ancient, non-symmetrical organisation, butsecondarily re-symmetrised its locomotory, outer body walland, to a large extent, nervous system as an adaptation to anew lifestyle. The developmental substrate of the previousnon-symmetry was however retained, for further elabor-ation of its increasingly complex viscera. Viewed in this waythe vertebrate body plan is a secondary re-imposition ofbilaterality, in the form of an almost symmetrical locomotor-skeletal outer body wall and paired brain structure, upon aradically non-bilateral adult body plan in the precursoranimal (see references cited in Section III.2). According tothis scenario, that non-bilaterality is evolutionarily deep,characterising a clade embracing most or all deuterostomeanimals. It was itself a secondary departure from originalbilaterality, the mode of axial organisation ancestral to allmetazoans other than sponges, cnidarians and possiblyctenophores (the clade Bilateria : Finnerty & Martindale,1998; Holland, 1999). Initial symmetry ‘breaking ’, invertebrate left-right development, is then seen as the reliablere-evocation of the ancestral adult non-symmetry, within thesymmetrical cellular anatomy of the contemporary blastula/gastrula. On this view, in most vertebrate embryos, therelatively recently imposed locomotory/neural bilateralityhas come to dominate the structure of the pre-gastrula socompletely that the lack of mirror-symmetry inherent inbiomolecular structure – usually referred to as molecular‘chiral ’ information – must be recruited to initiate theancestral gene cascades as what is now a left-right ‘axis ’.Tantalising indications do exist, however, of a left-rightcomponent at the outset of vertebrate development (seeSection III.2 and references).

II. OVERVIEW OF THE DEVELOPMENTAL

MECHANISM FOR VISCERAL SITUS

Fully detailed reviews of this expanding field are widelyavailable (see e.g. Tamura, Yonei-Tamura & Izpisua-Belmonte, 1999; Burdine & Schier, 2000; Capdevila et al.,2000; Schneider & Brueckner, 2000; Wright, 2001; Yost,2001; Hamada et al., 2002). Section II.1 inevitably involvessome detailed consideration of anatomy and moleculardevelopmental genetics ; readers interested in the widerissues might follow the remaining material without thissection, especially by referring to Fig. 1.

(1 ) A vertebrate-conserved ‘phylotypic’post-gastrular sector

It has become clear that activation of the transforminggrowth factor b (TGFb)-related intercellular signal gene

Evolutionary origin of vertebrate left/right asymmetries 379

‘Activin’signal

FGF8

BMP4Shh

Vgsignal

nodalsignal

leftysignal

flow atnode

Pitx2

(prolonged, extensive‘left encoding’)

BMP4signal

SnR

(’right encoding’for heart loop

& embryo torsion)

Shh

BMP4

BMP4

lateral

SnR

Pitx2

nodal

Fig. 1. Vertebrate left and right gene activity cascades. A generic embryo midline is represented as a ‘primitive streak’ or elongatedsite of gastrulation as seen from above, with the axis being generated in an anterior (top of page) to posterior sequence from aregressing node. Time progression is also represented from the top to bottom of the page. Thus the later and more lateral (‘phy-lotypic ’ – see text) gene expressions actually occur in post-gastrular structures, anterior to the level of a node more regressed thanthat shown. The earlier, near-midline ones in the chick occur around the node, before the onset of regression or during its earlieststages. Heavy hatched arrows connecting bolder gene names represent positive control (activation) input ; light stippled connectionsleading towards obliquely slashed gene names indicate negative control input onto their expression. Where the target of negative

380 Jonathan Cooke

nodal, in a broad domain in the left lateral mesoderm, is anessential conserved mechanism imbuing this region with leftidentity in all studied vertebrate embryos (Levin et al., 1995;Collignon, Varlet & Robertson, 1996; Lohr et al., 1998).Gastrulation proceeds as a sequence, in which mesodermdestined for successively more caudal regions of the bodyplan moves into place as a middle cell layer ahead of theclosing blastopore (in amphibians) or regressing Hensen’snode (avian and other ‘blastoderm type’ and mammalianembryos). Left lateral nodal expression initially follows thiswave-like sequence, beginning with material lying within orjust posterior to the heart-forming region. The mRNAexpression, presumably followed by peak levels of nodalsignal, is dynamic and is down-regulated after relativelybrief periods at successive antero-posterior levels, to disap-pear at a level near the regressing node after around 10somites have segmented, for instance, in the chick. Thisnodal signalling in turn activates transcription of Pitx2,encoding a homeobox-containing transcription factor. Onceactivated, left Pitx2 expression is both more durable thanthat of nodal and spreads anteriorly and posteriorly beyondthe nodal domain that triggered it. Pitx2, whose left-right roleappears to be that of an executive control gene for Leftdevelopmental character and tissue contribution to success-ive organs, is ultimately expressed throughout the deriva-tives of the left contribution to the heart tube, with a sharpboundary maintained from the inflow (future atrial) regionup into the outflow or ventricular region until advancedstages of looped heart morphogenesis. Over an extendedperiod as the body plan is laid down, Pitx2 expressionbecomes activated at the origin of the left contribution tosuccessive paired or asymmetrically developing visceralrudiments, such as those of lungs, stomach and otherintestinal derivatives (Logan et al., 1998; Piedra et al., 1998;Ryan et al., 1998; St Amand et al., 1998; Yoshioka et al.,1998; Campione et al., 1999).

The snail-related zinc-finger transcription factor gene,SnR in chick (Isaac, Sargent & Cooke, 1997), msna in mouse(Sefton, Sanchez & Nieto, 1998), becomes activated in right-lateral splanchnic mesoderm during gastrulation, in adomain that also appears to extend and then move back ina wave-like manner, but beginning slightly earlier than thatfor left-lateral nodal. Thus having begun in the anteriorlypositioned, compressed cardiac territory, right lateral SnRexpression is last detected in the posterior heart inflowregion only, at around the 12 somite stage in chick. Theorthologous snail-related gene is known from the frog (Xenopuslaevis) and zebrafish (Danio rerio), though expression studiesthat would reveal a distinctive right-lateral expressioncomponent have not been published. It thus seems reason-able to suggest that right-lateral expression of this genebe considered as an additional conserved component ofthe gene cascade for vertebrate situs. It should be noted

that nodal, Pitx2 and this snail-related gene each haveother, conserved but bilateral expression domains in earlyvertebrate embryos. These expressions, some of whichhave attested developmental roles, are under separatecontrol (see Isaac et al., 1997; Patel, Isaac & Cooke, 1999,for SnR).

It appears that where transient RNA expression isappropriate, as for the wave of nodal signalling, this isachieved through an early self-activating component in thetranscriptional control, followed by a damping negativecontrol input from a co-activated or downstream-activatedgene product. Thus a sequence motif in the control region ofnodal suggests positive feedback (i.e. self-activation) fromsignalling through the nodal receptor pathway itself, whilenegative feedback occurs through the antagonistic activity ofthe closely related ligand (or anti-ligand) lefty-2/antivin onnodal signalling at the protein level (Adachi et al., 1999;Norris & Robertson, 1999; Saijoh et al., 2000). The lefty geneis expressed in a left-lateral domain that closely tracks andslightly follows the nodal one, and is probably activateddirectly by the nodal signal that it then goes on to quench.Prolonged and stable expression such as is required forthe Pitx2 role, by contrast, is organised through initialupregulation by the upstream nodal signal, followed by self-maintaining and collateral positive maintenance inputs thatinclude a binding site for the Nkx 2.5 transcription factor(Shiratori et al., 2001).

Initial activation of lateral mesodermal SnR (chick) isbilateral, and could be considered a default state. Then withvariable rapidity among individuals, it is downregulated onthe left as nodal expression first appears there (Isaac et al.,1997). Experimental right-sided nodal protein expressionablates normal SnR expression there, while antisensetreatment targetting SnR expression ultimately leads toabnormal Pitx2 expression on the right (Patel et al., 1999),suggesting that SnR normally helps to suppress Pitx2expression there. Thus, while it is clear that left nodalsignalling positively activates Pitx2 transcription directly(Shiratori et al., 2001), a further, indirect double-negativecontrol relationship may help to ensure spatial exclusivitybetween the key snail-related and Pitx transcription factorgene expressions normal to right and left sides.

Pitx2 null mutant mice exhibit right cardiac isomerism,i.e. ‘ right-hand’ developmental character in heart precursortissue from both sides of the midline, although the situationfor more posterior viscera is less clear and may simplycorrespond to a lack of lateralising function, causingheterotaxia (Kitamura et al., 1999; Lin et al., 1999).Targetted ablation of msna in mice is not informative sincethe gene has other, prior essential developmental roles, butchick embryos after prolonged antisense disruption of SnR,having shown bilateral Pitx2 expression in heart precursors,then exhibit left-isomerism of heart-tube morphogenesis. In

control is itself an intercellular signal, control can be at the protein function rather than gene activity level. Lateral Vg (left) and BMP4(right) signals, placed at a horizontal dashed line representing the transition from early (near-midline) to ‘phylotypic ’ stages, areprobably phylotypic, while the evidence that the right-lateral SnR (snail-related zinc-finger transcription factor) role is phylotypicremains incomplete.


the same experiments, antisense-mediated SnR disruptionthat is too brief to cause detectable right Pitx2 expressionnevertheless randomises the direction of heart-tube looping.This looping direction, normally to the embryo’s right, is thefirst vertebrate-conserved, gross-anatomical manifestationof situs to develop. Loop reversal in these latter embryosis independent of the development of ‘ left ’ and ‘right ’structural character in the tissue contributions to the tubeinner wall, which can remain normal (Patel et al., 1999).Overall results of perturbing SnR function thus suggest adual role in left-right development. A direct role ensuresright cardiac looping and perhaps correct embryo torsion,but this is separable from the gene’s ‘cascade’ role inpropagating right-hand character within tissue by ensuringrepression of Pitx2. Pitx2, by contrast, appears to have aprolonged and widespread role, determining left-specificityin tissues of successively developing organs.

A variety of data demonstrates that in addition tonegative regulatory linkages between ‘opposing’ genes,barrier functions within the axial midline structures consti-tute a major mechanism maintaining proper laterality ofexpression domains normal to left and right during post-gastrular development (Meno et al., 1996; Bisgrove, Essner& Yost, 1999, 2000; Meno et al., 1999). The cellularstructure of the midline appears on anatomical groundsalone to constitute a relative barrier to diffusing intercellularprotein signals ; the notochord, a rod of tightly packedvacuolated cells with a tough matrix sheath, is tightlyassociated in vivo with the overlying floorplate, a midlinestrip of cells integrated into the neural plate but displayingat early stages several molecular affinities with notochord.There are also midline-associated gene expressions thatappear to have a ‘barrier ’ role, notably including a memberof the nodal-related lefty group (see above), whose proteinsmay act as an antagonist ‘ sink’ to nodal signalling by amechanism equivalent to ‘dominant-negative’ interferencewith nodal ligand. A lefty is expressed in a strip at the left-hand edge of, or bilaterally in, midline structures of allvertebrates examined. Disruption or non-formation of ananatomically normal midline, whether as part of a mutantphenotype or due to microsurgical or other early embryomanipulations, predisposes embryos to heterotaxias withbilateral or absent expressions of nodal and Pitx2 (Danos &Yost, 1995; Collignon et al., 1996; Tsukui et al., 1999).Several authors have classified anomalies of left-rightstructure and downstream gene expressions in relation tothe probable timing and axial position, within post-gastrulardevelopment, of midline barrier interruption (e.g. Bisgroveet al., 2000). Additionally, proper negative feedback onto theearly, autocatalytic activation of lateral nodal expressionappears necessary for its normal left restriction. Thus in amouse lefty null mutant, heterotaxias are associated withabnormally extensive and bilaterally spreading nodal RNAexpression (Meno et al., 1999; Hamada et al., 2002).

As with developmental ‘master control genes ’ generally,it has thus far been hard to make progress identifying targetgenes whereby Pitx2 (left) and the snail-like orthologues(right), actually execute the modulations of morphogenesisand growth rate that constitute ‘ left ’ and ‘right ’ charactersin relevant tissues. As regards heart loop morphogenesis,

there is evidence that differential microfilament functionand character of the extracellular matrix are involved(Itasaki et al., 1991; Tsuda et al., 1996). One complexity isthat structures that come to be situated at right and left inthe heart rudiment after its looping, were originally specifiedon the basis of relative antero-posterior ordering within thestraight heart-tube. Thus gene expressions characterisingparticular parts of the formed heart, that might beconsidered as members of specifically right or left develop-mental gene cascades, might in fact owe their left- or right-positioning more directly to correct looping direction,for instance via right SnR expression. A role for retinoidsignalling has been identified in the stable maintenance ofthe conserved post-gastrular left-right gene expressions, andthus the development of situs (Chazaud, Chambon & Dolle,1999; Zile et al., 2000), though this retinoid role does notappear to extend back into the earlier, symmetry-breakingphase that will now be discussed (Chen et al., 1996).

(2) Initial left-right symmetry breaking in earlierdevelopment: apparent diversification in timingand in upstream cascade steps

A sector of vertebrate development beginning late inneurulation, and extending through the organisation ofpharyngeal arches and a heart tube, can be regarded as arelatively conserved ‘phylotypic ’ anatomical stage, thepharyngula (although see Richardson et al., 1997). By thisstage, the vertebrate-conserved left-right gene expressionsdescribed above have become established in lateral meso-derm, and they too can justifiably be referred to as‘phylotypic ’. But vertebrate cleavage, blastula formationand early gastrulation are much more variable, and untilrecently, it has seemed that mechanisms of breakingsymmetry to initiate the phylotypic left-right cascades onthe correct sides may be as diverse, among vertebrateclasses, as is this earlier developmental anatomy. Compara-tive information for this early sector is with few exceptionsconfined to the widely used ‘model ’ embryo types : thezebrafish (Danio rerio), the clawed frog (Xenopus laevis), thechick (Gallus gallus) and the mouse (Mus musculus), plusrelevant human clinical genetic observations. Since this isessentially one embryo per taxonomic class, with noagnathans, elasmobranchs or reptiles, we cannot even besure that the differing timings of symmetry-breaking andfirst lateralised gene expressions observed typify the respect-ive classes ; such arrangements might be labile on an evenfiner evolutionary scale.

The various versions of early development seen inmammals are clearly among the most derived to be foundin vertebrates. Observations on early events in the mousewill nevertheless be described first, since they seem toillustrate most clearly the theoretical predictions that, if theembryo is initially of bilaterally symmetrical cellular struc-ture, left-right patterning must be derived by ‘conversion’from some form of chiral (i.e. structurally ‘handed’ inhaving no plane of bilateral symmetry) molecular infor-mation (Afzelius, 1976, 1985; Brown & Wolpert, 1990;Brown, McCarthy & Wolpert, 1991; Almirantis, 1995). Thediffering arrangements in the avian (chick) embryo will then

382 Jonathan Cooke

be described. Chick not only enjoys the longest list to date ofgenes with left-right lateralised expressions en cascade, butwas the first organism in which the recent explosive growthof molecular understanding began (Levin et al., 1995, 1997;Isaac et al., 1997; Pagan-Westphal & Tabin, 1998). Thischick gene cascade extends back into earliest gastrulation orbefore, and is accompanied by a consistent asymmetricalanatomy at mid-gastrulation (Wetzel, 1929; Lepori, 1969;Hara, 1978; Cooke, 1995). Incomplete pictures of earlyevents in fish and frog leave it currently unclear whetherthey align more closely with chick or with mouse in theirearliest lateralised gene expressions. This state of affairs hasbeen proposed to indicate a genuine evolutionary oppor-tunism or lability among vertebrate embryo types, in theinitiating mechanisms that precede the conserved ‘phylo-typic ’ sector (Yost, 1999, 2001; Wagner & Yost, 2000). Suchevolutionary lability of ‘upstream’ cascade steps in relationto a conserved, downstream sector in developmentalmechanism is seen elsewhere, for instance in sex determi-nation, and possible reasons for its origin have beendiscussed by Wilkins (1995, 2002).

Brown & Wolpert (1990) most clearly characterised therequirements for a molecular source of left-right symmetry-breaking information in an otherwise symmetrical embryo.This would have the formal properties of, for instance, theupper-case letter F. If molecules or molecular assemblies ofthis type were to become aligned in cells, with respect to twoother dimensions of organisation or ‘axes ’ that the cellsshared, a third dimension of alignment would necessarilybe created. If the initial shared alignments were antero-posterior and apico-basal, as could be the case in epithelialstructure near the midline in a gastrulating vertebrateembryo, then the molecular basis could be initiated for anorganisational difference between cells to either side of themidline, or for a left-right polarised transport via gapjunctions and the joint intracellular space. If an axis of beatof embryo cilia were to become fixed at an angle to anembryo-wide axial polarity shared by the epithelial cells,through a tethering according to the ‘F-molecule ’ principleof the known chiral molecular structure of ciliary basal bodycross-section, then net bulk transport towards the right orthe left of an anteroposterior midline axis in the epitheliumcould in principle result. Alternatively, a chiral (spiral)ciliary action with a particular (clockwise or anticlockwise)‘handedness ’ could be determinedmolecularly.With certainspatial arrangements in groups of such cilia near a midline,net bulk transport to the right or left could conceivablyresult, although in this latter case it is important to recognisethat the F-molecule principle is not involved, since individ-ual ciliary assemblies are only aligned with respect to theone, apico-basal cell polarity. The general principles ofembryo symmetry-breaking by ‘conversion’ from chiralmolecular structure, together with one idea for their possiblerealisation via ciliary activity, are illustrated in Fig. 2.

It was proposed many years ago that normal ciliaryactivity in the embryo might somehow be translated into thecorrect directionality of visceral situs, based on the inheritedhuman Kartagener’s syndrome. In these individuals, ab-normal ciliary cross-sectional structure at the electron-microscopic level, and paralysis of ciliary beat, is linked with

a random incidence of visceral situs inversus and situs solitus(Afzelius, 1976, 1985). All known cilia/flagellae in the bodystructure, including spermatozoa tails, are affected in thisparticular syndrome, causing infertility and chronic airwaycongestion and infection as additional aspects. But structur-ally atypical cilia, the protocila, have long been known toexist in early vertebrate embryos, distributed one per cell onthe apical surfaces of epithelia facing certain cavities. Theirmotility and functional significance had been questionable,but following a recent exciting series of reports, it is clearthat such cilia are indeed motile. Gene products that arenecessary for their structure and normal motility are alsodemonstrably required for normal symmetry-breaking insitus development, at least in mammals.

A densely distributed group of protocilia on the ventralsurface of the mouse embryo node (anterior tip of theprimitive streak), creates in vivo a net flow toward the leftacross the embryo midline (Nonaka et al., 1998). Normallythis must occur within the narrow extracellular space that isthe homologue of the primitive vertebrate archenteron. Thisoccurs during the middle part of gastrulation including thestart of node regression or head process formation. Inmouse, the midline notochord population derived byingression at the node is at first included in the epithelialsurface of the future foregut roof, and for some timeprotocilia remaining on these cells continue densely topopulate the emerging midline anterior to the regressingnode. These cilia may also function to maintain the leftwardflow or, alternatively, may contribute to left-right barrierformation (see Section II.1). Ciliary activity somehowcreates net flow to one side of the midline.

A variety of targetted null mutant mice, lacking functionof particular proteins involved in ciliary construction andactivity, show either absence or paralysis of the embryonicprotocilia in conjunction with gross disturbances of down-stream ‘phylotypic ’ left-right gene expression and visceralsitus. One of these corresponds with the previously known iv(inversus viscerum) mutant, the gene product now beingidentified and characterised as left-right dynein (LRD), arelative of the ‘axonemal ’ dynein subfamily (Supp et al.,1997, 1999). In both mutant versions, where stiff, paralysedprotocilia are reported though other cilia are normal, thepredominant phenotype is random assignment of embryosto either normal or near-normal situs, or complete or near-complete situs inversus, with correspondingly reverse-later-alised expressions of nodal, lefty and Pitx2 genes (Okada et al.,1999). See Fig. 1 for the normal gene expression cascade.Other mutants, for proteins integral to protociliary mor-phogenesis or activity (Nonaka et al., 1998; Marszalek et al.,1999; Takeda et al., 1999) and for the forkhead transcriptionfactor HNF4 (Chen et al., 1998), have no recognisableprotociliary structures. In these, interestingly, heterotaxiasmay predominate, with absent or abnormal bilateralexpressions of ‘phylotypic ’ left-right genes, rather thannormal or reversed ones.

These correlations between ciliary activity and its knownmolecular basis, the demonstrated extracellular ‘nodalflow’, and symmetry-breaking for visceral situs in a mam-mal, have undoubtedly impressed the community as a majorinsight into mechanism. They indicate that reliable net


Fig. 2. Possible conversion modes from molecular chirality to left-right developmental information. Upper two diagrams illustratehow a macromolecule or molecular assembly showing chirality (properties formally similar to the letter F – see Brown & Wolpert,1990), initally present as randomly orientated copies in the cells of an epithelium having apico-basal structure and an already-developed axial or antero-posterior polarity (open arrow), could achieve alignment by means of anchoring within cell structure. If,for instance, the cells were linked by gap junctions, such a molecular assembly could mediate directional intracellular ‘morphogen’

384 Jonathan Cooke

leftward transport of an intercellular signal, initially pro-duced symmetrically around the midline, initiates thedifferential cascades of the lateralised gene expressions.Once started correctly, these are maintained as exclusive bytheir regulatory cross-relationships and by the midlinebarrier function (see Section II.1). Since the nodal gene isalready locally active around the base of the regressing node,and indeed in the mouse this transcription normallybecomes progressively enhanced on the left edge (Collignonet al., 1996), a direct working hypothesis would have nodalprotein itself as the leftwards-translocated ‘morphogen’ (seeHamada et al., 2002). This idea has the added attraction thatregulatory relationships between nodal and its immediatelydownstream and related gene lefty appear to embody theprinciples of local self-activation (by nodal protein) leadingto longer-range lateral inhibition (by lefty protein). Thesehave long been proposed on theoretical grounds to charac-terise symmetry-breaking morphogen systems, whose be-haviour when computer-modelled can amplify a small initialdisturbance or inhomogeneity into a major, stable asym-metry in the spatial distribution of a developmental signalconcentration (Turing, 1952; Gierer & Meinhardt, 1972;Gierer, 1981).

Nonaka et al. (2002) now claim to have demonstrated thedirect action, in mouse at least, of fluid flow across therelevant embryo surface in determining the directionality ofthe ‘phylotypic ’ gene expressions and of situs itself. Thestrikingly ingenious experiments involved culture of theembryos in a microchamber, in which medium flows ofcontrolled rates could be imposed from either left to right orright to left in relation to the primary embryonic axis. Theflow rates that reversed situs of normal embryos, or coulddetermine that of ivx/x embryos (with inactive cilia), weregrossly comparable with those caused by the normal ciliaryactivity. Interference was most effective in the periodimmediately preceding normal onset of left-lateral nodalgene expression. At the same time, the authors emphasisethe incompleteness of our understanding of the physicalbasis of the endogenous directional flow, which appearsliterally to cross the field of beating cilia rather than, forinstance, being whirlpool-like with a particular ‘handed-ness ’ around the node. If the beat of individual protocilia issimply spiral, the net leftward flow would need to resultsomehow from their dense distribution coupled with theantero-posteriorly distinctive shape of the node base. In thisway chiral molecular structure with only one axis ofalignment, the apico-basal epithelial axis, could be con-verted to left-right directionality at a tissue level without

recourse to the ‘F-molecule ’ principle. If, on the other hand,flow caused by each individual cilium has net directionalitybecause its beat has an off-axial in addition to a spiralcomponent, as speculated above, the ciliary basal bodystructure could indeed be embodying the full ‘F ’ principle ofBrown & Wolpert (1990; see also Brown et al., 1991,discussion transcript). The structure might be tethered,within each cell, in relation to some embryo-wide anatom-ical co-ordinate in addition to the apico-basal cellular one.

In birds, the blastula-stage embryo is essentially a sheet ofcells that are developing epithelial structure, within which abar-like thickening due to the piling up or ‘columnarisation ’of cells comes to define an axis, the primitive streak. As inthe mouse, the definitive postgastrular midline arises as cellsingress through the shortening streak to form the newendoderm and mesoderm layers, the space beneath defini-tive endoderm ahead of the node being equivalent toprimitive archenteron. These arrangements have obviousstructural homology with those in the mouse, but the chicknode is a less focussed structure and built on a more massivecellular scale. The mouse ‘ turret ’ structure with clear upperneurectodermal and basal endodermal epithelial layers (seeBellomo et al., 1996) is not found. As gastrulation enters thenode-regression stage in chick, the notochord occupies anew middle layer from its inception, without transientintegration into the midline archenteron (foregut roof).Single protocilia exist on many cells over an extensive,diffuse region near the midline, throughout gastrulation, onthe apical (i.e. outer) surfaces of both endodermal andupper, neurectodermal layers. They are sparsely distrib-uted in space on endoderm due to the flattened, stretchedand heterogeneous nature of these cells, and this situationpersists once node regression has started (Manner, 2001;Essner et al., 2002; J. Cooke, unpublished observations).This renders implausible the production of significantdirectional flow as seen in the mouse, especially if ciliarybeat has a spiral component. The apical neurectodermalprotocilia in this central region are somewhat moreprominent, but it should be recognised that any structural‘handedness ’ within the anchored molecular assembly ofcilium and basal body itself, when viewed from a particularsurface of the embryo, would necessarily be reversed asbetween the apical cell surfaces of endoderm and neurecto-derm. To suppose anything else would be to deprive thismolecular assembly of the crucial ‘conversion’ role (Brown& Wolpert, 1990) that is the whole attraction of a proposedassociation of ciliary action with the origin of tissue levelleft-right asymmetry.

transport (arrow from left to right) to initiate differences across the axial midline. Lower digrams illustrate two different ways inwhich chiral structure inherent in cilia at the basal surface of an embryo midline region, shown facing the reader, could create netflow of extracellular medium to the embryo’s left (reader’s right), as in the observations of Nonaka et al. (1998). In the left diagram,individual cilia express chirality only as the ‘handedness ’ of their spiral mode of beating and are thus not anchored in twodimensions within cell structure according to the ‘F-molecule ’ principle. Net directional flow results in some way from co-operativitybetween such cilia in relation to the antero-posterior architecture of the embryo’s node region. The right diagram shows thesituation where the chiral structure within individual cilia allows for anchoring at a particular angle to the embryo’s long axis, so thattheir intrinsically directional, whiplash-like beat produces the net leftward flow. It remains unclear, at the time of writing, whichprinciple is most used in the real situation.


In addition to the above problems, a peri-nodal flowcomparably timed with that seen in the mouse would beredundant as an initiator of lateralised gene expression inthe chick. A cascade of such expressions, preceding anyknown for mouse, bridges between the earliest gastrula oreven blastula stages and the onset of the ‘phylotypic ’ nodal/lefty/Pitx2/SnR cascade (Levin et al., 1995, 1997; Boettger,Wittler & Kessel, 1999; Rodriguez-Esteban et al., 1999;Yokouchi et al., 1999) (see Fig. 1). Thus, while cilia areindeed diffusely present on the chick nascent archenteronsurface, around the time that lateralised gene expressionsare first detected in early gastrulation, these associatedlateralised gene expressions are quite different from those inmouse (e.g. Collignon et al., 1996; Lowe et al., 1996).

Chick activin receptor IIA mRNA is first detected preferen-tially on the right flank of the early primitive streak, thenopposite the anterior streak to the right. This occurs beforethe appearance of definitive node structure or regression,and thus in the absence of any structural midline barrier.Since this particular receptor is known in other circum-stances to be inducible by an appropriate ligand, a priorstep in the cascade is probably the right-sided accumulationof such a ligand, perhaps by preferential gene expression.Selectivity relationships between known receptors andligands within this superfamily are incompletely known, sodespite a report of preferential right activin bB mRNAexpression at the same early stages (Levin et al., 1997), itremains unclear that the ligand involved has been identified.Experimentally, activin protein perturbs downstream left-right gene expression and situs when applied to chickblastoderms during these early stages (Levin et al., 1995;Isaac et al., 1997), but experiments may not have beensufficiently controlled to ascertain that perturbation distinc-tively follows from a left-hand experimental application.Recombinant activin protein is highly diffusible, and as willbe seen, further roles for other ligands of the TGFbsuperfamily, in addition to the well-understood one of nodalitself, have been postulated for both right and left sides.

In all vertebrates, the signalling gene sonic hedgehog (Shh)has a prominent expression (and role) in the new post-gastrular mesodermal and neural midline that emergesahead of the regressing node. Early on, in chick, thisexpression usually appears more prominent in the presump-tive neural ventral midline (floorplate) than in underlyingchordamesoderm. Soon, it becomes significantly extendedback into the node wall on the left side only. Experimentsindicate that it is the preferential right-activation of the‘activin pathway’, just described, that normally repressesShh expression to the right of the node. Chick nodalexpression arises shortly afterwards in deeper-lying, meso-dermal structure immediately lateral to the Shh expression inthe left node, and this clearly corresponds with the left-accentuation within the bilateral ring of nodal expression atthe base of the mouse regressing node. Though this near –midline left nodal expression is maintained for some hours inboth types, no cascade role for it is evident experimentally inchick. Instead, the left-lateral Shh signal at the node directlyevokes the separate, broad left-side ‘phylotypic ’ nodalexpression via a double-negative mechanism, involvingactivation of the Cerberus-Dan related extracellular signal

gene caronte (Rodriguez-Esteban et al., 1999; Yokouchi et al.,1999; Zhu et al., 1999). Caronte protein is thought tocounteract, on the left, the otherwise widespread bilateralbone morphogenetic protein (BMP)-4 signalling at thisstage. This in turn relieves a pre-existing BMP-4 repressionon nodal expression, thus involving yet another member ofthe TGFb superfamily in left-right determination.

This early, near-midline chick cascade appears locked in,via reciprocal repressive relationships, with another main-tained just to the right of the midline. Thus the right-accentuated ‘activin pathway’, in addition to repressing Shhexpression at the right in the node, activates fibroblast growthfactor (FGF )-8 expression in the right anterior flank of thestreak behind the node (Boettger et al., 1999; Schneider et al.,1999). FGF-8 signalling in turn can repress Shh, andactivates the transcription factor gene NKX3.2 in the rightstreak flank as well as the right-lateral ‘phylotypic ’ SnR (seeSection II.1). The potential complexity is indicated by thecapacity of BMP-4 signal, whose gene is intensely activatedat right of the normal node, to repress normal Shh andactivate ectopic FGF-8 expression when locally applied atleft (Monsoro-Burq & Le Douarin, 2001). Additionally, ataround the time of these differential Shh, FGF-8 and BMP-4expressions, the specific adhesion-mediating molecule N-cadherin is expressed in an asymmetrical pattern around thenode and anterior streak (Garcia-Castro, Vielmetter &Bronner-Fraser, 2000). Role relationships are not yet clear,but interestingly, effects of blocking N-cadherin function onheart-loop direction and embryo torsion may be mediatedwithout affecting the otherwise key downstream ‘phylo-typic ’ Pitx2 and SnR expressions. This raises the possibility ofparallel independent, and not interlocked, gene expressioncascades.

The role of the right-preferential ‘activin-pathway’expression, at the head of the known chick cascade to date,may be a more direct reflection of an initial symmetry-breaking process than is the subsequent cascade of later-alised expressions. Following activin protein applicationbefore stages of node regression, doubly right-sided, doublyleft-sided, left-right reversed and normal versions of thedownstream gene expressions are produced in approxi-mately equal numbers of embryos (Isaac et al., 1997). Thissuggests failure of symmetry-breaking followed by a recov-ery process, in which repressive regulatory links ensure thateither ‘right ’ or ‘ left ’ cascade but not both gets activated,independently on the two sides but otherwise at random.Such an outcome is reminiscent of those resulting from theinactivations of LRD or ciliary kinesins in mouse (Collignonet al., 1996; Nonaka et al., 1998; Marszalek et al., 1999).

It has been hard to establish whether embryo typesrepresented by zebrafish and Xenopus laevis fall more into linewith mouse, in proceeding directly from a symmetry-breaking mechanism to the phylotypic cascade, or withchick in utilising an intermediate cascade of lateralised but‘near-midline ’ gene expressions. Attempts to investigatethis in zebrafish through microinjection of Shh-expressingor -interfering constructs are hard to interpret, since thegene has two potential roles in left-right development. In allvertebrate embryos it supports normal function of thenotochord and floorplate and thus of the midline signalling

386 Jonathan Cooke

barrier, in addition to any left-specific signalling role it mayhave. Thus in mouse, where no functional or expressionevidence for a left-specific Shh role exists, the Shh null mutantphenotype is now recognised to show left-right anomaliesconnected with breakthrough expression across the midlineof the ‘phylotypic ’ nodal and Pitx2 expressions (Meyers &Martin, 1999; Tsukui et al., 1999). To date, no evidencesuggests that the cascade just described in chick, beginningwith right-hand accentuation of an ‘activin pathway’ andproceeding through left midline Shh expression via a caronte-like function to the left phylotypic cascade, is utilised by frogor fish.

We have seen that the derivation of left-right organisationfrom chiral structure (Fig. 2), i.e. symmetry-breaking bymolecular ‘conversion’ (Brown & Wolpert, 1990) occursconsiderably earlier in chick development than does the‘nodal ciliary flow’ that is proposed to initiate this in mouse(Nonaka et al., 1998). The mystery intensifies in that, bycontrast, such cilia occur in Xenopus laevis (frog) and Daniorerio (teleost fish) embryos only at late gastrular or even‘pharyngula ’ stages, and at posterior axial positions (Essneret al., 2002). Especially in the fish case, the timing andposition seems hard to link with an origination of thelateralised gene cascades through archenteron-located flow.

(3 ) Is there a unitary mechanism fordevelopmental origin of vertebrate situs?

Further problems of interpretation remain, for the simplestview of a vertebrate-universal, initial symmetry-breakingmechanism via leftward ciliary flow. The iv gene productLRD, despite its family relationships, is largely distributedcyoplasmically and in regions outside those where activeprotocilia occur, including an anatomically left-lateralisedcomponent, in the gastrula/neurula (Supp et al., 1999). Sucha distribution would not be consistent with a role confined toinitial symmetry-breaking by molecular conversion. Thepossibility exists that LRD, and/or some of the other geneproducts whose requirement for normal symmetry-breakinghas been assumed to follow from their functions in ciliumformation/activity, have additional functions in polarisedtransport machinery within the joint (gap-junction linked)intracellular space. These transport functions, directionallytethered within cells on the ‘F ’ principle (Brown & Wolpert,1990), could also be relevant to symmetry-breaking bymolecular ‘conversion’. An explanation may still berequired for the apparently different consequences of LRDloss on the one hand (random situs correlated with paralysedcilia) and loss of other proteins such as kinesins on the other(heterotaxia and absent cilia), although a larger databasewould be desirable.

Of most difficulty is a mouse mutant not yet mentioned,inv (inversion of embryo turning (Yokohama et al., 1993;Lowe et al., 1996), that almost completely reverses situs atpopulation level rather than randomising it or causingheterotaxias. Elucidation of the nature and cell function of aprotein whose mutational alteration achieves such a reversalhas been eagerly awaited, during the gene’s protractedpositional cloning since its initial characterisation. ‘ Inversin ’turns out to be a large, intracellular protein of unclear

relatedness, containing a domain of multiple ankyrin-typestructural units believed to signify tethering to other proteins(Mochizuki et al., 1998; Morgan et al., 1998). The mutantphenotype also exhibits early lethality due to kidneymalfunction, possibly signifying inversin involvement intranscellular transport mechanisms (see reference below toCa2+ signalling), but characterisation of the cellular func-tion remains elusive.

Crucially and paradoxically, inv null mutant mouseembryos do not exhibit reversed nodal ciliary flow. Instead,a possible subtle alteration in shape of the node base, and asomewhat more turbulent flow of reduced efficiency inleftward transport of experimental microparticles are re-ported (Okada et al., 1999). The authors of the nodal flow-based hypothesis of symmetry-breaking (Nonaka et al.,2002), understandably, have attempted to modify theirmodel to incorporate and explain this finding, but at the costof depriving the model of its original merit, which was as anexplanation for de novo symmetry-breaking. The modelmight nevertheless be rescuable from these paradoxicalobservations if, for instance, it were ultimately to turn outthat some intimate cellular architecture of the basal node ornearby midline was instrumental in the normal leftward netflow. The inv mutant alteration, a partial deletion within theprotein structure, might conceivably alter this in a way thatfailed to reverse the experimentally observed flow but didreverse an in vivo functional equivalent. It must be pointedout, however, that the published report of manipulations ofthe flow does not include results of the formally appropriate‘negative control ’ condition, namely a manipulated absenceof net flow (Nonaka et al., 2002). Such controls areparticularly desirable in view of the known tendency ofculture in flowing-medium conditions to randomise situsdevelopment in mouse and indeed in other vertebrateembryos (Brown et al., 1991; Fujinaga et al., 1992; Fujinaga,Lowe & Kuehn, 2000; J. Cooke, unpublished observationson chick and blastocoel-irrigated Xenopus laevis embryos).

Meanwhile, evidence has accumulated for the role ofdirectional intracellular transport via gap junctions, insymmetry-breaking at the earliest stages of both chick andXenopus laevis development (Levin & Mercola, 1998a, b,1999; Levin et al., 2002). Experimental interference withgene expression for a connexin that is a principle compo-nent of embryo gap junctions, and related pharmacologicalchannel interference, results in downstream randomisationof situs and associated gene expressions in both species. Anintercommunication barrier is reported to exist, for bothfrog blastula and chick blastoderm, at positions that could betaken as ventral within the respective fate maps. Under thesecircumstances, if transport machinery had been aligned on‘F-molecule ’ principles in relation to the embryo axis,preferential ‘morphogen’ transport could occur across apresumptive dorsal midline to build up differences betweenright and left. The linking of frog and chick embryo types byevidence of this sort is significant because they appearotherwise divergent, within the range of more downstreamcascade steps, since the latter but not the former exhibits thenear-midline sequence of local gene expressions that startsearly in gastrulation and culminates in left Shh and Caronte(see Section II.2). Observations on mouse, where the claim


for causal primacy of ciliary flow seems strongest, furthersupport a generality of the involvement of intracellular smallmolecule transport in the symmetry-breaking phase for situs(Pennekamp et al., 2002).

A final recently forged link between apparently divergedembryo types concerns the distinctive left-right role of the veg(vg) genes, encoding yet another subgroup of the TGFbsuperfamily of signal proteins. Vg1, the first-identifiedmember in Xenopus laevis, has been a strong candidate foran endogenous maternally encoded signal whereby theyolky vegetal blastomeres induce mesoderm and definitiveendoderm in the equatorial zone of the blastula (Thomsen &Melton, 1993; Kessler & Melton, 1995). Orthologues andclosely cognate genes are now known additionally from frog,zebrafish, chick and mouse (Seleiro, Connolly & Cooke,1996; Sun et al., 1999; Wall et al., 2000). Their candidacy forprimary axial patterning roles has perhaps been usurped bythe nodal genes themselves (Varlet et al., 1997; Varlet,Collignon & Robertson, 1997; Brennan et al., 2001), even inthe yolky eggs of frog and fish where maternally translatedgenes are prominent at early stages (Schier & Shen, 2000). Adevelopmental patterning role for Vg genes is neverthelesssuggested by their special processing requirements forrelease of active ligand. The enzyme system, wherebyfunctioning C-terminal polypeptide is cleaved off from thepre-proprotein at secretion, is not the widely available oneutilised by TGFb family members generally. Normaldevelopment in the frog and the chick occur withoutproduction of immunologically detectable amounts of theworking Vg ligand, despite abundant bilaterally distributedmRNA and pre-proprotein. The assumption is that theendogenous role(s) of Vg genes involve localised productionof the minute amounts of ligand required, due to localisationof a crucial part of the processing machinery.

Experimental ectopic mis-expression of Vg genes employschimaeric DNA or RNA constructs, in which the C-terminal distinctive Vg sequence is fused with an N-terminalsector encoding the permissive processing parts fromanother TGFb gene. In the frog such experimental mis-expression has been found to exert a distinctive effect on situs(Hyatt, Lohr & Yost, 1996; Hyatt & Yost, 1998; Hanafusaet al., 2000). Applied on the right, Vg is able systematicallyto reverse the expression of the ‘phylotypic ’ cascade andmorphological situs itself, rather than randomising these orgiving rise to left-isomeric development as experimentalright expression of nodal or Pitx protein would tend to do.There is additional molecular evidence that the experimen-tal effects are distinctive to Vg itself, rather than due todirect right-ectopic activation of the response pathway tonodal protein, for instance. This has led to a proposedendogenous role as a ‘ left-right co-ordinator ’ signal innormal development. The implication is that this role isnormally exerted from a relatively lateral left position. Theendogenous time of action is unknown, but data suggest thatexperimental action on situs is still fully effective in the earlygastrula stage, and possibly much later than this (Toyoizumi,Mogi & Takeuchi, 2000).

Either chick Vg, or chick or mouse nodal proteins havebeen locally mis-expressed on the right, in an extensiveseries of chick blastoderms in culture ( J. Cooke &

S. Withington, unpublished work). Here, too, Vg exerts adistinctive effect. Whereas Nodal protein, present on theright from stages of node regression and midline formation,randomises situs and induces right ectopic expression of nodaland Pitx2 genes as would be expected, right-expressed Vgprotein is able with significant frequency to reverse theexpression of Pitx2 to give right expression only. Further-more, as would be expected from its downstream position inthe normal cascade, right-ectopic nodal protein is unableever to disturb the left-sided Shh expression at the node itself(or the associated gastrular structural asymmetry, seeSection III.2). Comparable ectopic Vg protein expressiondoes indeed disturb or reverse these features, when imposedfrom stages before they have developed. Finally, the nullmutant phenotypes have been described for the mouse Vg1orthologue Gdf-1 (Rankin et al., 2000), and for furin, aconvertase component of a processing pathway that maydistinctively be required for certain TGFb superfamilyligands (Constam & Robertson, 2000). These involve rightisomerism and left-right inversions of particular visceralarrangements, and typically, absence of the ‘phylotypic ’left-lateral gene expressions. These observations all suggestthat Vg genes have a vital vertebrate-conserved role, situatednot far downstream of the initial symmetry breaking processfor situs.

(4) Changing left-right co-options of geneorthologues during vertebrate diversification,and other puzzles

FGF-8 in chick has a distinctive right-hand expression whichis functionally integrated into that species’ early near-midline cascade (see Section II.2). The evidence in mouse,however, is that it is a left-specific player at comparablestages (Meyers & Martin, 1999). The transcription factorgene Nkx 3.2, a left-hand player apparently downstream ofnodal and kept repressed by right-hand FGF-8 in chick,correspondingly appears to swap sides in mouse (Schneideret al., 1999). In chick, as also described in Section II.2, theextracellular caronte protein is crucially involved in propa-gating left information from the node into lateral mesoderm,being induced by left Shh and acting to de-repress phylotypicleft-lateral nodal expression by interfering with BMP-4signal. In mouse, where Shh has no known lateralisedexpression or role and where a left caronte gene expressionorthologous with that of the chick has not been found, agene cognate with caronte, Dte, is nevertheless reported toshow distinctive right-hand expression at comparable stagesin the node (Pearce, Penny & Rossant, 1999). This ispuzzling for an additional reason; potentiation, rather thancounteraction, of BMP-4 signal is believed to be importantat a right-lateral position, as this antagonises Vg signal there(see above, Ramsdell & Yost, 1999; Branford, Essner &Yost, 2000). Most tested Cerberus-Dan family proteinsantagonise BMP-4 signalling although dte has not explicitlybeen tested.

In any version of a lateralising gene cascade, there is noreason why a particular gene should not have distinctiveroles in different steps on opposite sides of the body, pro-vided that the cascade steps are sequential, circumscribed in

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time and well-regulated within the developing anatomy.BMP-4, for instance, might have more than one successiverole on the right, counteracting Vg signalling from the leftin all embryo types (Ramsdell & Yost, 1999; Branfordet al., 2000), and restricting Shh expression at the node to theleft in types utilising this expression (Monsoro-Burq & LeDouarin, 2001). Additionally, it has a later one on the leftin connection with the heart in at least one embryo type(Chen et al., 1997). It is partly for this reason that negativeinterference or targetted mutagenesis of receptor anddownstream signal transduction components can be prob-lematic in interpretation when there is a ‘ left-right ’ pheno-type. Each deleted component may have had normal rolesthrough signalling by more than one ligand within asuperfamily, and could thus have acted in ‘right ’ or in ‘ left ’propagation, or in symmetry breaking, at different times. Arecent report concerning FGF-8 in rabbit establishes thatapparent ‘ side-swapping’ of gene roles between embryotypes can occur even within a vertebrate class (Fischer,Viebahn & Blum, 2002), and may be more dependent uponthe morphology of gastrulation than upon deeper evolution-ary change. This paper also clearly indicates the interpret-ative perils attending multiple successive roles for particulargenes within one overall ‘cascade’.

Despite striking apparent differences, observations cur-rently leave room for the belief that both the left-rightdevelopmental gene cascade and the underlying mechan-isms of deriving symmetry-breaking information from chiralmolecular structure, might be conserved for vertebrates.Protocilia probably are present, albeit in a wider and lessdense distribution, across the archenteron or equivalentsurfaces of all vertebrate embryo types over a prolongedperiod of early development. Pending an exact understand-ing of the way in which these cilia operate, the possibilityremains of their broadly conserved role involving directionalextracellular transport. But there is the strong additionalpossibility that some early process of intracellular polarisedtransport is universally present, at pre-gastrula or laterstages, and functionally important in symmetry-breaking forall vertebrate types. There is a need to elucidate the role ofthe inversin (inv) protein. Additionally, a process at or closeto the head of the cascade also appears universally to in-volve the left-lateral Vg signalling, while other TGFb signalsuperfamily members have widespread roles. It is note-worthy that this superfamily of signal proteins, centrallyinvolved in vertebrate axial induction and anteroposteriororganisation (Smith et al., 1990; Cooke, 1991; Schier &Shen, 2000; Brennan et al., 2001), should be central also inthe mechanism of situs.

III. VIEWPOINTS ON THE EVOLUTIONARY

ORIGINS OF SITUS

(1 ) The functional ‘ just-so’ story: situs as avertebrate (chordate) invention

It can plausibly be argued that, provided a basis for itsmechanism existed within cellular organisation, a reliably

directional packing of the major viscera within the stream-lined body wall would have been strongly selected for duringthe origin of modern vertebrates. The argument runs asfollows. Transition from a sedentary or crawling, detritus- orfilter-feeding, to an actively swimming predatory lifestyleis likely to have accompanied the origin of the vertebrateclade. Greatly increased metabolic demands would havebeen involved. Additionally, a square–cube relationshipapplies between available surface areas for nutritionalabsorption and gas exchange on the one hand, and tissuemass on the other, if strictly equivalent anatomical structureincreases in linear size. If, as seems likely, a progressive sizeincrease took place, there would therefore have been strongselective pressure for proportional increase in the lengthsand complexity of tubular viscera and of pumping vascu-lature. Within the streamlined outer ‘ locomotory’ bodyshape that was meanwhile being selected for, such increasesfor rate of food absorption and of vascular exchange couldonly have been accomodated by the more complex loopingand/or coiling of these organ systems. For gut function, therelationship is intuitively obvious, while in the case ofvascular function the effect of a spiral and looped arrange-ment, in optimising the pumping output per unit massof muscular tube wall, has recently been described for themammalian heart (Kilner et al., 2000). Provided that thevisceral packing problem is ‘solved’ in precisely the sameway in all normally developing individuals, the furtheropportunity arises for intimate co-ordination, thus subtleco-optimisation, between the originally quite independentperistaltic actions of vascular and digestive systems.

Given a bilaterally symmetrical cellular structure of thegastrula, and the disparate origins of the internal organrudiments within the body plan, regularisation of internalpacking could only occur if cascades of gene expressions,propagating separately in tissues to either side of themidline, imbued them with left and right positionalidentities. In this way, left-right differential growth andmorphogenetic patterns could be initiated co-ordinately inthe various internal rudiments. The mechanistic develop-mental problem appears as that of the reliably directional, or‘handed’ breaking of the initial embryonic bilateral sym-metry, and its conversion into the initiation of the lateralisedgene expression cascades. As detailed in Section II.2,Brown & Wolpert (1990) made an influential formulation ofthe problem in asserting that the only mechanism wherebysuch directional symmetry-breaking could occur is by ‘con-version’ from the information implicit in a chiral macro-molecule or molecular assembly. It is widely recognised thatindeed some such molecular conversion process is needed ifthe embryo starts out bilaterally symmetrical at the cellularlevel of structure. Thereafter, appropriate regulatory exclus-ivities between the lateralised gene expressions that aretriggered, together with a stabilising barrier function in themidline against cross-invasion of signals, will ensure geneti-cally ‘ left ’ and ‘right ’ sides with potentially separatelyevolvable developments.

Among vertebrate embryo types examined to date, themajority indeed appear entirely bilaterally symmetrical atstages before the first features of situs develop. The bilaterianclade of animal forms, by definition, is considered to have


Fig. 3. Schematic of a proposed dexiothetic transition within vertebrate ancestry. Three body forms are shown, each in dorsal planand in composite transverse sectional views (-axial levels indicated by dashed lines). The original, truly bilaterian ancestor on the lefthas paired special sense organs (black) and filter- or detritus-feeding apparatus anteriorly, paired and probably segmented mesodermstructures (stippled), and a tubular gut without left-right but possibly with dorso-ventral complexity. The central diagrams representthe body form shortly after the morphological transition, whereby an original right side became a ‘ventral ’, substratum-applied side,

390 Jonathan Cooke

derived from a common ancestor having acquired an axis oftrue bilateral symmetry. Most recent accounts of theexplosion of molecular knowledge about vertebrate left-right development have, understandably, been from mol-ecular genetic researchers whose project is a mechanisticunderstanding of contemporary embryonic development.They have tended to lack exposure to the disciplines ofcomparative anatomy and phylogenetics, with their accenton the dimension of historical contingency in evolution andtheir propensity to ask: ‘via which route do currentbiological arrangements happen to have come about? ’ Ithas therefore been easy to propose or assume that asymmetry-breaking mechanism, and the gene-expressioncascades for situs, were ‘ invented’ entirely in response to theselective pressures attending the ancestry of vertebratesthemselves, or at least that of the clade of most obviouslyrelated organisms in which they are embedded, namely thechordates. The exciting correlation of mutations that affectciliary structure or function with disturbance of situs and ofthe ‘phylotypic ’ left-right gene cascades in mammals (seeSection II.2), has brought the feeling that an understandingof this molecular ‘ invention’ is close at hand. The wholemechanism seems to conform with what theory woulddictate as necessary, during each contemporary vertebrateembryo’s development, for directional de novo breaking of a‘primordial ’ bilateral symmetry.

(2 ) A constrasting view: situs as the co-optationof a pre-existing axis

There exists a substantial alternative viewpoint to the above,however, based on comparative anatomy and phylogenetics.This would not postulate that the ur-vertebrate or even ur-chordate started out with axial symmetry of the bilateriansort. Rather, it is seen as having evolved a secondary,derived bilaterality in the outer ‘ locomotory’ body wall andnervous system, in relation to the newly active mode of life.Enclosed within this bilateralised shell was an archaic‘visceral ’ body that had, much earlier in the history ofdiversification of animal forms, abandoned the primordialsymmetry ancestral to the Bilateria in a profound morpho-logical transformation. In essence, the ancestral bilaterianright-hand side is proposed to have become a new ‘ventral ’surface applied to the substratum, with resultant loss ofprimary bilateral symmetry to give a sessile or slow-moving,detritus- or filter-feeding form. This proposed transform-ation, perhaps with unfortunate consequences for its sale-ability to a wider community, has been termeddexiothetism. It may have occurred with the foundation ofthe entire clade known as the Deuterostomia, or alterna-tively, be shared by a more restricted grouping that includes

echinoderms and chordates (see below). Vertebrates wouldon this view retain a deeply embedded, non-symmetrical‘visceral ’ body organisation resulting from the dexiothetictransformation, combined with an axial but only secondarilyre-symmetrised ‘ locomotor ’ component. The archaic com-ponent including its regionalised developmental gene ex-pressions, laid out in a defined way with respect to this newlyre-symmetrised organisation, would provide the basis forany retained, then further elaborated, internal left-rightstructure. Dexiothetism is illustrated in outline in Fig. 3,together with its proposed consequences for vertebratestructure.

Hemichordates, echinoderms, urochordates, cephalo-chordates and vertebrates rather clearly form a clade, thenow-accepted Deuterostomia, when fossil (stem) forms,anatomical structures shared by extant forms, features ofembryonic development and the newer (mostly 18S ribo-somal DNA-based) molecular systematic evidence are allconsidered together (Gislen, 1930; Jefferies, 1975; Jefferies,1986; Jefferies, 1991; Jefferies, Brown & Daley, 1996;Cameron, Garey & Swalla, 2000; Holland, 2000). TheHox-type homeobox gene cluster seems basal to animalaxial organisation, and the possession of a relativelycomplete cluster of Hox gene orthologues even by echino-derms, that have no apparent axial bilaterality in theirextant life-histories, justifies the assumption that the deuter-ostome clade shares a true bilaterian ancestry (Peterson,Cameron & Davidson, 2000). Current attempts at molecu-lar phylogenetics within this clade lack resolving powerhowever, such that almost every group named above canappear to be a basal sister group to all the others if particulargenomic sequences are considered alone !

One possible scenario is thus that hemichordate adultbilaterality is indeed of the original bilaterian kind, and thatthis was then abandoned at the origin of a clade including allthe other groups (e.g. Jefferies et al., 1996). Nevertheless,certain shared echinoderm, hemichordate and urochordateasymmetries of coelomic openings, together with the torsionevent that attends settling at metamorphosis by larvalechinoderms (see e.g. Ruppert & Barnes, 1996, pp. 923–924), suggest that what becomes an ‘oral-aboral ’ axis inadult echinoderms has somehow become left and right againin adult forms of the latter two groups, which thus havesecondarily re-acquired an adult bilaterality after its loss as aprimary character. These animals in turn share enoughdistinctive anatomical structures to link them indubitablywith the cephalochordates (or acraniates : Amphioxus etc.) andvertebrates. This is consonant with the overall best-supported DNA sequence-based phylogeny proposed byCameron et al. (2000), which places hemichordates as sistergroup to echinoderms, and these together as sister group to

with concomitant invasion of ventral specialisations and loss of some subset of the original right members of paired structures.The result is a non-symmetrical body plan. The right-hand diagrams show an arbitrary stage in the secondary re-acquisition ofbilaterality in sensory-locomotory structure (by organ-pairing anteriorly, and perhaps progressive re-rotation and forward invasionof the locomotory tail structure with a notochord). This allowed essential symmetry of the outer body wall together with a mor-phological or molecular developmental basis for left-right structure centred in the viscera. This scenario takes account of evidencefrom comparative developmental anatomy of extant forms, comparative gene expression and molecular taxonomy, but does notfollow any one detailed phylogeny derived from fossil evidence.


all the rest (see also Castresana, Feldmaier-Fuchs & Paolo,1998). Hemichordates would then have shared in theancestral loss of primary bilaterality, in which case theyappear secondarily to have re-acquired bilaterality morethoroughly than any other deuterostomes. On this scenariothe developmental left-right asymmetries of gene expressionand anatomy in chordates, which are bizarre and gratuitous-seeming for Amphioxus and tunicate tadpoles (see below),but underlie the situs adaptation to the high-energy lifestylefor vertebrates, can all be seen to be recapitulating a non-bilateral ancestral phase.

To be of relevance to vertebrate origins, the dexiothetictransformation would have to have occurred by theCambrian. It is furthermore important to distinguishbetween the basic proposal of dexiothetism outlined above,for which there is considerable supporting evidence ofdiverse kinds, and on the other hand those detailedcladograms that have been proposed, based on structuralinterpretations of post-Cambrian calcichordate fossils, fortransitions defining echinoderm, urochordate, acraniate andvertebrate ancestry (e.g. Gislen, 1930; Jefferies, 1975, 1986,1991; Jefferies et al., 1996). Evolutionary developmentalbiologists inevitably see the anatomical interpretation offossil traces as extremely subjective and fragile, in compari-son with the kinds of data they are accustomed to. Criticalappraisal of the plausibility of the dexiothetism proposal perse, as relevant to the origin of vertebrate left-right structure,should probably not turn on these detailed scenariosfascinating to specialist systematists though they are. Afurther liability is that dexiothetism has been suggested tohave occurred essentially within a single ancestral individualthat, having begun life as a conventional bilaterian, founditself flattened onto its right-hand side and made the best ofit. While indeed imaginable, this ‘creative monster ’ scenariofor the transformation is not a necessary one, and does notsell it well to most contemporary evolutionists who aregradualists. In reality, a dexiothetic transformation couldhave been as gradual as was any other innovation in theCambrian.

The extant life history giving the best glimpse of what wasinvolved may be that of a crinoid echinoderm, whereby thelarva’s right coelomic system atrophies or never develops,while the left one becomes ‘upper ’ or circum-oral in theadult after the final torsion movements of settling (Holland,1991). Other extant echinoderm classes have, probablysecondarily, inverted this arrangement at metamorphosis.While some forms (holothuroids and burrowing echinoids)re-acquire very superficial forms of bilaterality, all share afundamental pentaradial symmetry around the adult oral-aboral axis. It has recently become apparent that a sector ofthe echinoderm Hox gene array, corresponding to moreposterior axial levels of other bilaterians, is bilaterallyexpressed, with ‘colinearity ’ along the larval coeloms beforethe settling and torsion events whereby original right and leftcoelom sets become superimposed to give the oral-aboralaxis (reviewed in Popodi & Raff, 2001). The crinoid settlingtransformation is equivalent to a dexiothetic one (assumingretention of mouth and feeding apparatus on the originalleft, now ‘dorsal ’ side), and thus may be a primitivecharacter within echinderms. An alternative scenario for

loss of primary bilaterality in deuterostome phylogeny, theconverse of dexiothetism, has also been proposed (Holland,1988). That one or other of these scenarios approximateshistory is more probable, on the basis of all the evidence tohand, than that the basis of situs is a chordate innovationsuperimposed on retained primary (-‘ur ’) bilaterality. Aproposal whereby a primary ‘bilateral ancestor ’ directlygave rise to extant pentaradial echinoderms (Morris, 1999)seems lacking in plausibility, quite ignoring the morphologi-cal transitions and torsions of metamorphosis.

Fossil forms that are contentiously classified either asechinoderms or as calcichordates did not acquire penta-radial adult symmetry, and include perhaps the leastsymmetrical or indeed ‘axial ’ forms known within theBilateria (e.g. Cothurnocystis, illustrated in Jefferies, 1991 andJefferies et al., 1996). It is from among these that the extanturochordates, cephalochordates and chordates are proposedto have derived, through sequences of steps involvingprogressive symmetrisation of the head (feeding structuresand sense organs) and in some lineages, re-acquisition offorward adult motility followed by an ‘ invasion’ of a re-symmetrised locomotory ‘ tail ’ into the more dorsal partof the head. Satisfactory demonstration of relatively ‘sym-metrised’ chordate characters in a calcichordate fossil,whose diagnostically echinoderm-type skeleton in turn linksit with the likes of Cothurnocystis, is important for theplausibility of the scenario. Thus the recent contributiondescribing paired gill slits in Jaekelocarpus is noteworthy(Dominguez, Jacobson & Jefferies, 2002), but will doubtlessnot end the controversy.

What follows is based, then, on the contentious but Ibelieve broadly well-founded proposal that chordates have amorphological transformation in their ancestry whereby aprimordial dorsal-ventral organisation became a right andleft one. Importantly, there are no grounds for suspectingthat either the tunicate (urochordate) or Amphioxus (cephalo-chordate) body plans have undergone regression from anyphase supporting vertebrate-like activity levels, and thusrequiring complexity of visceral packing (see Section III.1above). Indeed, while Amphioxus lacks a heart, presumablysecondarily and in relation to small body mass and inactivelifestyle, it shares with adult tunicates some simple left-rightgut structure as well as asymmetries within the branchialfeeding apparatus. From this point of view, it can be seenthat the ‘evolution’ of gene cascades to characterise left andright body sides of a proto-vertebrate would almost certainlyhave begun from the preservation of a pre-existing geneticarchitecture. Jefferies has daringly suggested that the currentleft-right regionalising gene expressions of chordates shouldbe, or at least prominently include, orthologues of the ur-bilaterian dorso-ventral set as known from the fruit flyDrosophila melanogaster. However, as Gerhart (2000) haspointed out, our expectations regarding the conservedregionality of developmental gene expressions may be naivein relation to the depth of evolutionary time. The transitionsand divergences under consideration may have occurredamong animals whose cross-sectional anatomy, at least, wasless tightly organised than in extant forms.

The problem of the reliably directional breaking ofbilateral symmetry during ontogeny of each contemporary

392 Jonathan Cooke

vertebrate embryo can be seen as no different from that ofthe reliable ‘ settling ’ of an echinoderm larva on oneparticular side to form the oral-aboral axis of the adult.Close observation shows that in at least some such larvae,the latter axis has already come to be prefigured beforesettling, as left-right differences in the openings betweencoelomic cavities and in archenteron-associated left-right(future oral-aboral) gene-expression asymmetries (McCain &McClay, 1994; Hardin, 1995). Molecular analysis of thesignalling mechanism that orientates the site (and polarity)of adult rudiment development within the larva (see Aihara& Amemiya, 2001) will obviously be of great interest. Invertebrate gastrulae, most of which appear completelysymmetrical at the level of cellular anatomy (though seebelow for discussion of the bird embryo), it may be thatthe re-symmetrised locomotory component of adult structurehas come to dominate development, to the point where allgene expression or egg-structural cues for initiating the left-right (pre-dexiothetic dorso-ventral) axis are temporarily lostduring each ontogeny. Access to macromolecular structuralchirality is then used to resurrect the organisation of thisaxis, and an epigenetic mechanism of access to suchinformation, using ion-channel-mediated vectorial intra-cellular transport and/or ciliary-mediated flow (SectionII.2), might therefore be a genuine chordate/vertebrate‘ innovation’ even if left and right gene cascades are not.Alternatively, such a mechanism could have been there allalong, and utilised within developing deuterostome larvae ofmany groups to ensure directional metamorphosis or‘ settling ’. A conserved mechanism that turned out to utiliseciliary action would be understandable, in view of the smallsize and prominent deployment of cilia in locomotion byplanktonic larvae. In many ciliated deuterostome larvae,including Amphioxus as well as sea urchins, locomotion isspiral in a way conditioned by the structurally tethered ‘off-axis ’ beat of the cilia, and not solely by macroscopicmorphological chirality that the larva happens to show(reviewed in Chia & Buckland-Nicks, 1984).

There exist, however, a few tantalising indications ofdeep-seated left-right asymmetry associated with the veryonset of development, or with the foundations of axialpatterning as such, in urochordate and vertebrate de-velopment. In holoblastically cleaving frog and ascidianurochordate eggs, the first cleavage plane usually has a closerelationship with the ultimate midline plane of the body. Yetleft-right differences have been found that indicate eccentriclocalisation of certain ion-channel-mediating gene productswithin these early blastomeres (Albrieux & Villaz, 2000;Levin et al., 2002). This would indicate a left-right componentin the reorganisation movements that localise the sources ofaxial inducers, best understood in the frog egg, and theremay be a link here with the asymmetry of Veg-relatedfunction that is probably an early conserved step in the left-right cascade (see Section II.3 and elsewhere). The otherindication of the primacy of left-right structure in chordatedevelopment may be the avian gastrular asymmetry.

All three species of avian embryo that have beensufficiently investigated, chick, quail (order Gallidae) andduck (order Anatidae), show a pronounced, consistent buttransient morphological asymmetry in the mid-gastrula

embryo during the first few hours of node regression(Wetzel, 1929; Lepori, 1969; Hara, 1978; Cooke, 1995).While we cannot interpret this asymmetry directly in termsof the recapitulation of any known ancestral anatomy, thatmay be the most parsimonious explanation for it. It appearsfunctionally gratuitous, and long precedes the subtle left-right fate-map, growth and morphogenetic asymmetriesthat execute the first functional aspect of situs itself, namelyheart-tube looping. Since it will be familiar to few readers, adiagram of this asymmetry appears in Fig. 4.

During the developmental period concerned, the wings ofcolumnarised epithelium flanking the node are the pre-sumptive diencephalic and midbrain neural folds, with theprosencephalic (presumptive cerebral) territory lying aheadin the midline. The midline mesoderm emerging fromthe node during the period of the asymmetry embraces theregion of the prechordal-notochord boundary and thenotochord of hindbrain-cervical levels. This period ofgastrulation/neurulation begins slightly before the peaks inintensity of left-hand Shh expression extending into the wallof the node, and of right-hand accentuation of FGF-8expression in the streak (Dathe et al., 2002), though it doesnot precede the initial lateralised gene expressions of theavian blastoderm (see Section II.2). A gap develops, in theotherwise continuous wall of deeply columnar cells accumu-lating at the node, where the epithelial structure is thinnerand thus forms the root of a ‘gutter ’ in the neurectodermalsurface. The gutter extends forward as the floorplateprecursor, or midline of the future neural tube. During theasymmetry, this gap is not at the anterior face of the nodeand thus the tip of the shortening streak, but rather, alongthe left node wall.

Shh expression in the midline is strongest at these earlystages in the presumptive neural floorplate, rather than thenascent notochord beneath. It thus marks the emergence ofnew floorplate cells around the node’s left side. The deeper-lying nascent chordamesoderm is derived entirely from theanterior and right-hand side of node structure during thisperiod. Thus the right-derived notochord root and left-derived floorplate root are markedly offset in plan view ofthe embryo, only swinging into line to form a unified axialstructure some time after the regressing node has recededfrom them in the gastrulation process. Morphologies of theleft and right wings of the neural plate, and of the underlyingshoulders of paraxial mesoderm, are also markedly andconsistently different at this stage. Later, from around thetime of the fifth somite segmentation onwards, they attainsymmetry, and notochord and floorplate origins becomesuperimposed in the midline of the now symmetrical node.

When account is taken of the fact that much peripheraltissue in the bird blastoderm is extra-embryonic in fate, theasymmetry can be seen as quite massive and central withinthe axial body plan. The bird gastrula of this stage, viewedwithout any pre-conceptions derived from the bilaterality ofthe later vertebrate body, does not appear remotelysymmetrical. Furthermore, the asymmetry appears gratu-itous in relation to any possible later differences, in relativeamounts of tissue assigned from left and right, to paired butasymmetrically developing structures such as the heart tube.Even the greatest functional asymmetries of the avian brain


(see Section IV) are never accompanied by left-rightdifferences in neural tube wall morphology or thicknesscommensurate with what is transiently seen during neuralplate formation. Only some 12 h later does the heart tube,forming in the midline from paired contributions that weremuch more lateral in the gastrula, swing and loop to theembryo’s right as the first conventionally recognised ana-tomical mark of situs development.

This handed morphological asymmetry has been univer-sal in some hundreds of relevant-staged normal embryos inthe author’s accumulated laboratory observations, togetherwith correlated asymmetry of Shh expression in all caseswhere revealed by in situ hybridisation. It occurs even if axial

development has been otherwise perturbed and delayed byexplantation from the egg into culture at much earlierstages. This extreme stability of the gastrular handedasymmetry contrasts with the lability, attributable to theex ovo culture, whereby up to 10% reversal in situs itself(heart-loop and embryo torsion directions) is subsequentlyseen. Experimental manipulation of the postgastrular‘phylotypic ’ gene expressions, that themselves postdate thefirst development of the gastrular asymmetry, effectivelyrandomises situs as described in Section II. Developmentclearly proceeds through factors that robustly set up thegastrular asymmetry from an apparently bilateral earliercondition, then on through subsequent steps whereby

A B

C

Fig. 4. The asymmetrical anatomy of the avian (chick) gastrula. (A) The whole embryo-forming area (area pellucida) of the blasto-derm is shown from the dorsal surface, anterior at top. Dashed box indicates the central region, embracing the centre of gastrulationactivity (Hensen’s node) together with the base of the emerging postgastrular midline (head process). (B, C) Detail of boxed regionin A from dorsal aspect (B) and in transverse section (C). The axial level of section is indicated by a dashed line in B and the observerof the section faces anteriorly. Arrows indicate indentations in the neurectodermal surface at the edges of the thickened node.The left indentation is much deeper, and continuous both with the central pit of the node via a gap in the node wall, and with thegutter-like groove that passes anteriorly to swing into the midline as the future floorplate of the nervous system (all shown cross-hatched). The right-hand indentation flanks the deep-lying mass of the nascent notochord, which is positioned eccentrically to theright. The emerging notochord swings progressively into place in the midline beneath the floorplate (boundaries indicated in stipple)as the node regresses from each level.

394 Jonathan Cooke

‘phylotypic ’ situs-related gene expressions are normallyinitiated in more lateral mesoderm. These subsequent steps,while they may normally lie downstream in cascade fromthe ‘gastrular asymmetry ’ and associated near-midline geneexpressions, are distinctly less robust against developmentalperturbation. It should be recalled that the asymmetries ofcell membrane electropotential found in chick early streakstage blastoderms also predate the structural asymmetry(Section II.3). Presumably these are robust in culture, atleast acutely, or they could not have been studied (Levin &Mercola, 1998b, 1999; Levin et al., 2002).

Only two experimental circumstances in chick have everbeen observed to reverse or obliterate the structural mid-gastrular asymmetry. One is early exposure to experimentalperturbations of ‘ the activin pathway’ or to Vg protein,another TGFb superfamily ligand with a probable veryearly vertebrate-conserved left-right role (see Section II.3).The other is the occurrence of twinned pattern within singleblastoderms in a way that causes separate but parallelregressing nodes. In this latter case, specifically the right-hand of the two nodes tends to be perturbed or reversed forthe asymmetry while the left one remains robust. Inresponding to conjoined twinning according to this particu-lar rule, the gastrular asymmetry aligns with visceral situsitself and with the habenular/parapineal neural asymmetry(see Section IV.3), as first revealed in classical amphibiantwinning experiments (Spemann & Falkenberg, 1919;Oppenheimer, 1974; see also Levin et al., 1996).

Any comparable structural asymmetry could have goneundetected in the small and often transparent gastrulae offish. Amphibian gastrulae on the contrary are opaque, andthe structure equivalent to that which shows the avianasymmetry may be internalised. The relative inaccessibilityof the narrow relevant time-window, among the greatvariety of blastoderm-type telolecithal amniote (reptile)embryos, has prevented our charting the incidence of suchgastrular asymmetry among them. Though dramatic oncefocussed upon, it has scarcely been noticed or remarked onby whole generations of chick embryologists. Thus, apartfrom stating that it is not detectable in the very small scalecellular structure of the mammalian (mouse) regressingnode, we cannot currently assess this asymmetry as acharacter on any vertebrate cladogram.

In Amphioxus species, the bizarrely accentuated develop-mental asymmetries of the pharyngeal region and headcavities again seem gratuitous with respect to current larvalor adult function. It is thus difficult not to see these, alongwith the relative symmetrisation that follows, as some kindof recapitulation. The ultimately left set of pharyngeal poresor gill slits arises first, on the animal’s right, then migratesacross the ventrum to its definitive position, leaving thedefinitive right set to form later in situ. This sequence fitswith the notion of a calcichordate ancestor which had anon-symmetrical head with only one, upward-facing setof gill pores, but which subsequently underwent an organ-pairing approach towards symmetry (see Jefferies, 1991;Jefferies et al., 1996). Signs of ancestral non-symmetry in thelarval urochordate are more subtle, but are visible inthe asymmetrical dorsal pharyngeal structure and visceraof the adult.

Most recently, strong evidence in favour of the ‘co-option’ as against the ‘de novo ’ hypothesis, for the origin ofthe left-side vertebrate gene cascade, has come from the newdiscipline of ‘comparative molecular anatomy’ ; that is, thestudy of the expressions of orthologous genes in differentanimal types. Amphioxus nodal, Pitx and HNF3b orthologues,and urochordate (ascidian) nodal and Pitx orthologues, havenow been studied in this way (Terazawa & Satoh, 1997;Schumpert et al., 2000; Yasui et al., 2000; Boorman &Shimeld, 2002a ; Morokuma et al., 2002; Yu, Holland &Holland, 2002; reviewed in Boorman & Shimeld, 2002b).All have distinctive left-sided expression components duringembryonic/larval development that are clear homologues ofthe vertebrate ones. In the ascidian tadpole these expres-sions appear completely gratuitous, since the structure issymmetrical at this stage (though it could conceivably haveundergone secondary simplification). Interestingly, left-lateral hedgehog orthologue expression in Amphioxus is inter-preted as due to, rather than instrumental in causing, themarkedly asymmetrical head anatomy. The expressionconcerned is not the homologue of the Shh expression atleft of the chick node, but only of the much later anteriorendoderm expression in the vertebrate ‘phylotypic ’ stage,which is centred around the midline just as is the vertebratemouth and pharynx itself (Shimeld, 1999).

These asymmetries of gene expression, with the equallygratuitous-appearing asymmetries of anatomy, are presentin animals where neither are interpretable as adaptations tocurrent lifestyle features. Nor have we any reason to believethat these extant chordate relatives have ancestors that hadadopted the vertebrate ‘high energy lifestyle ’, with itsadaptive ‘need’ for right-left developmental gene marking.Yet the gene expressions are clearly homologous with thosein vertebrates, where they control the development ofanatomical asymmetry, namely visceral situs, to which wedo tend to ascribe an adaptive function. The situation asregards the right-lateral expression component of the snail-related gene is currently unresolved, as the orthologue isknown from Amphioxus (Langeland, Tomsa & Jackman,1998) but such an expression was not explicitly investigated.Its major role within vertebrate situs itself is almost certainlymore restricted than those of the left ‘phylotypic ’ geneexpressions however, perhaps being related only to heart-looping and embryo torsion (Isaac et al., 1997; Patel et al.,1999). It could thus have been a genuinely novel co-optionat the vertebrate transition in relation to the need for heartcomplexity.

IV. VISCERAL MAJOR ORGAN,

NEURO-ANATOMICAL AND

NEURO-FUNCTIONAL LEFT AND RIGHT:

UNITARY OR INDEPENDENT ORIGINS?

(1) Hominoid hand-use/linguistic lateralisation:origin and inheritance

Most people are familiar with the finding that, world-wide, perhaps just over one in ten human individuals


systematically prefers to use the left hand for most manipu-lative activities and shows correspondingly greater skill withit, whereas the remainder are ‘right-handed’. Today it isalso accepted that this ‘handedness ’ is fairly deeply embed-ded in most individuals’ development, rather than being theresult of education or social convention, though people canstill be found who assume the latter, especially withincultures that are strongly normative about the hand to beused in writing, eating or personal hygiene. In fact, mid-gestational foetuses appear preferentially to suck their rightor left thumbs in proportions corresponding with theincidence of later right- and left-handedness. Data fromsites of stone tool construction have moreover shown thatproportions of right- and left-handers have remained closeto contemporary values through the palaeolithic (Toth,1985), and may have exhibited pronounced right-bias fromthe inception of stone tool use, that is, before the emergenceof our own species.

A variety of gross anatomical asymmetries in the regional-ised structure of the cerebral cortices form a system thatis probably linked developmentally with hand-use prefer-ence. This system is certainly linked with the functionallateralisations whereby language and associated phenomenaare normally subserved by identified regions in the leftfrontal and parietal cortex, Wernicke’s and Broca’s areas(Geschwind & Levitsky, 1968; Galaburda, 1991). Theseareas tend statistically to greater development on the leftthan the right, an asymmetry that is reduced and morevariable rather than clearly reversed among left-handers etc.Since motor output from left cortex more immediatelycontrols the right side of the body, it can be said that in thesimplest instance, the left cortex and right hand form a unitthat expresses communicative and manipulative capacitiesthat most obviously distinguish our species. The corre-sponding (and perhaps further) brain regions on the rightsubserve complementary non-linguistic, emotional-spatialfunctions that are harder to define (Geschwind &Galaburda,1984; Bradshaw & Rogers, 1993; Corballis, 1997).Functional criteria for lateralisation have normally beenthe adequacy or otherwise of preserved function followingextensive damage or ablation on the respective sides. Theleft-right partitioning may be more a quantitative than anabsolute one for each neural network concerned, andassociated with preferential developmental neural ‘pruning’on the non-dominant side rather than extra production onthe dominant (Galaburda, 1991).

The precise relationships between brain-anatomical andfunctional aspects of this lateralisation system are complex,because while clearly correlated at the population level theyare imperfectly correlated within individuals (see e.g.McManus, 1991). These probabilistic relationships, togetherwith the generally greater directionality for the functionalthan for gross-anatomical asymmetries in humans (around10:1 as opposed to 7:3), have led to the suggestion that,evolutionarily, the language/hand-use system has beensuperimposed on a substrate provided by brain asymmetrieswhich may be more ancient. It is now known that at leastthree great ape species exhibit most of the gross anatomicalcerebral asymmetries equivalent to the human ones, thoughperhaps at somewhat reduced population directionality, and

with greater quantitative variability (e.g. Cantalupo &Hopkins, 2001). Until recently, directional hand-use asym-metry had not been thought to characterise ape populations,at least for tool use (though see Annett & Annett, 1991;Corballis, 1991). But it is now realised that at leastchimpanzees (Pan troglodytes), bonobos (Pan paniscus) andgorillas (Gorilla gorilla) may have two modes of hand use. Forone of these, that uniquely accompanies communicativevocalisations and may be termed ‘proto-linguistic ’, individ-ual lateralisation is found with a right preponderance thatappears to correspond with the incidence of greater leftdevelopment of the ape speech-area homologues (Hopkins& Leavens, 1998). Language thus appears to have its distin-ctive evolutionary roots quite deep within the hominoidclade, but may have capitalised upon a pre-existing tendencyto neural lateralisation that is essentially vertebrate-deep(see later discussion).

Variations in hand-use/linguistic functional lateralisationfollow a definable, even though statistical, pattern ofinheritance within the human population. Understandingthis is of great interest to our overall topic, even though itincludes as yet no molecular characterisation. The popu-lation and familial data are almost equally well explained bythe models of McManus (1985, 1991, 1995), and Annett(1985). These differ mainly in that the former assumes thatevery normal individual is at depth lateralised, and that thelateralisation (for hand use) is one of preference, with skilldevelopment as a secondary consequence, while the latterassumes a population continuum of intrinsic left-right skilldifferential, with a right-shifted mean. These models are notexplanations of the origin of biological information for thelateralised development ; rather, they assume its universalexistence, but postulate genetic variation in the efficiencywith which it is ‘ read’ in individuals’ developments. Dataare broadly consistent with segregation of two alleles at asingle locus, with semi-dominance such that one homo-zygote results in strong reading of the information, i.e. inessentially 100% right-handedness and ‘ left-(language)brainedness ’ at population level ; the heterozygote resultsin a reduced right-shift at the population level and thusin around 75% right-handedness etc., and the otherhomozygote results in no right-shift and thus in equalincidences of developmental outcomes, utilising epigeneticmechanisms following the failure of ‘genetic ’ access to theunderlying lateralising information. With appropriate allelefrequencies, involving a high though probably not 50%incidence of the ‘ loss of function’ allele, the population dataon incidence and inheritance of handedness are accountedfor. A simple dominant-recessive situation is precludedbecause pairs of left-handed parents, as a population, do notproduce as many left-handed as right-handed children.Thus, either they must include people who are hetero-zygotes, at least, for ‘right handedness-access ’, or elsehomozygosity for the ‘recessive ’ allele must still allowappreciable developmental bias towards right handedness.It is crucial to understand that no alleles ‘ for ’ reversal of thecommonest ‘ right-handed, left-brained’ situation are postu-lated, but only ones affecting efficiency of access tobiological lateralising information that will be present in allnormal embryos.

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The McManus model addresses the data on knownincidence of the various combinations of brain languagelateralisation and handedness, and accounts for them onlyby assuming that each genetic situation exerts its probabil-istic effects independently upon these two aspects offunction. Interesting small caveats are needed (and supplied)for the known greater incidence of left-handedness in males,while a certain aspect of data on identical twins needsindependent explanation (see Section IV.2). Though noeffects of these alleles upon Darwinian fitness need bepostulated under contemporary conditions, a polymorphismbalanced by long-term selection acting in favour of hetero-zygotes through human pre-history is indicated, if theincidences of genotypes have been as stable as archaeologi-cal evidence suggests. Annett & Manning (1989, 1990)make interesting conjectures, with some evidence, thatwould account for this, and could also explain the often-reported elevations from the general population incidence ofleft-handed individuals in certain occupational groups orworking environments. The explanation proferred is thatbeing strongly genetically right-shifted (a ‘strong right-hander ’) may place limits upon development of certaincognitive skills, selectively excluding such individuals (whowill tend to be ‘right-shift ’ homozygotes) from pursuit ofcertain occupations. But the existence of a degree of lateralpartitioning of complementary brain functions is assumedto optimise the capacity for complex behaviour (see e.g.Bradshaw & Rogers, 1993; Andrew, Tommasi & Ford,2000). A stable polymorphism involving an incompletelydominant allele that favours lateralisation would thereforeensure that at least in a numerous class of individuals, allthese lateralisations exist in one particular directionalrelationship, but are not overly extreme in degree.

(2 ) Hand-use/linguistic lateralisation andsitus in development

Clearly, the numerous human individuals with combina-tions of neural functional and gross cerebral lateralisationother than the commonest one do not all exhibit situs inversusor heterotaxias ; the latter conditions are rare ones. An obviousdevelopmental question is then: is the biological infor-mation, used with variable efficiency to set up the cerebralfunctional and gross anatomical neural lateralisations dis-cussed above, derived ultimately from the situs controlcascade discussed in Sections II and III? Or is it the result ofan independent cascade of gene expression, as yet unidenti-fied, which nevertheless is initiated by the same molecularconversion process of symmetry-breaking as that whichinitiates the situs cascades? A third possibility is that both theunknown gene expressions involved, and the symmetry-breaking process that initiates them, are quite separate fromthe corresponding situs-controlling ones in the case ofhuman neural functional laterality. Note that in the lattercase either the symmetry-breaking itself, or the mechanismthat interprets it developmentally, may contribute to therelatively reduced (70–90%) population-level reliability for‘normal ’ hand-use/linguistic laterality and its cerebralanatomical correlates, in comparison with the extreme(around 99.99%) reliability of situs direction.

An extensive study by Torgerson (1950), of all situs inversusindividuals in a large sector of the Norwegian population,has recently been extended with the inclusion of contem-porary neural functional imaging methods (Kennedy et al.,1999; Tanaka et al., 1999). By no means all human situsinversus cases correspond with mouse mutants where motorprotein lesions are associated with failure of an initialsymmetry-breaking step (Schneider & Brueckner, 2000).Nevertheless, the documented inclusion of a sizeable sampleof cases associated with the rare bronchiectasis/ciliary stasis(Kartagener) syndrome, means that lesions in such a step arealmost certainly represented in the ‘experimental ’ humanpopulation. It is rather clear that the 1 in 104 subset of viablehumans with situs inversus totalis exhibits a distribution of thecerebral functional lateralisations that is similar to that inthe general population, rather than being its ‘mirror ’converse. This striking finding appears to necessitate thatthe third possibility above is the case : that the two systems oflateralisation are developmentally quite independent. Fasci-natingly, one further gross-anatomical brain asymmetry,that tends to a 7:3 directional distribution in humans, alignsclearly with the situs asymmetry rather than with the hand-use/linguistic systems in these surveys. This is the thesomewhat greater occipital projection of the cerebral cortexwithin the skull on one side, and corresponding frontalover-projection on the other, known as petalia. Thus, whileany functional significance for this asymmetry is currentlyunclear, it can be considered a subtle manifestation of situs(see Section I).

There remains one paradoxical observation that, takenalone, would indicate a deep relationship between thedevelopment of human situs and functional neural lateral-isations. This is the elevated discordancy rate for bothfeatures, above statistical expectation, that exists betweenmembers of a particular small subset of identical (mon-ovular) twin pairs. These are the pairs whose pattern ofplacentation and amnion formation suggests a twinningevent that occurred late within the period that permits fullyseparated development of the embryos themselves withinthe multicellular conceptus (Griffiths & Phillips, 1976; Burn,1991). As was mentioned in Section III.2, an experimentalembryological observation is that major situs features, as wellas the habenular diencephalic and avian gastrular asym-metries, tend to reversal in the right-hand member of late-separated experimental twins. This might be expected if anearly lateralised intercellular signal, normally confined bymidline barrier formation, was to the right of the normalaxial midline. Then, in the right-hand member only ofcellularly conjoined axes, this ‘right ’ signal might abnor-mally be experienced by the left side, thus perturbing orreversing the situs cascades of that member. Such pertur-bation has now been observed, in appropriate spontaneouslytwinned chick embryo axes, at the level of expression oflateralised Shh and other genes (Levin et al., 1996; J. Cooke,unpublished observatons). From clinical observations offrankly conjoined (Siamese) human twins, it is clear that theright-hand member is predominantly at risk for situs-relateddefects. An assumption then is that among the separatelydeveloping but ‘at risk ’ twin pairs, it is the originally right-hand members who show the isolated (and in any case rare)


situs defects. Since neither right- nor left-lateralisation forneural function is rare like situs disturbance, it remainsless clear whether among twins discordant for these func-tions it is the ‘right ’ or the ‘ left ’ member who is liable toundergo lateralisation ‘reversal ’. Limited clinical data how-ever suggest that it need not be the member who has sus-tained situs perturbation (Burn, 1991). Thus the generalclass of mechanism for laterality disturbance following twin-ning may be similar in the situs and the functional neuralcases, namely abnormal cross-interference by lateralisingintercellular signals. But the above studies on large situsinversus samples indicate that the actual signals involvedare different, and independent, for situs and for hand-preference/linguistic function.

(3 ) Status of other vertebrate functionalforebrain lateralisations

During the last 10–15 years there has been increasingrecognition that left-right partitionings of forebrain func-tion, significantly directional at the population level, arewidespread among vertebrates of all classes where behav-iour has been intensively studied (see Bisazza, Rogers &Vallortigara, 1998; Andrew, 2000 for reviews). Theysubserve such diverse cognitive and behavioural systems asvisual discrimination learning (Gunturkuhn et al., 2000),approach-avoidance and aggression (Rogers, 1990, 1991;Miklosi & Andrew, 1999), imprinting-type learning (re-viewed in Horn, 1998), locomotory behaviour (Ross &Glick, 1981), bird tool construction and use (Hunt, Corballis& Gray, 2001), the organisation of bird song (Nottebohm,1971), and bird navigation (Wiltschko et al., 2002). In mostreports, unfortunately, the mode of data presentation doesnot reveal whether individual variation was more the resultof differences in degrees of lateralisation, or more due to anincidence of unlateralised or reverse-lateralised individualscontributing to pooled data. Examples where the strength ofdirectionality can be deduced, however, run from the barelysignificant (60%, for rat locomotory control), through the70–75% level with reversal rather than non-lateralisation ofminority individuals (pigeon differential eye use in discrimi-nation), to a level that equals or exceeds the 90+% thatcharacterises human language/hand-use (chick imprinting-related behaviours). Gross anatomical or neural structuralcorrelates are not usually known, though in some casespreferential isotope incorporation on one side is a majorevidence for lateralised activity (Horn, 1998).

There is as yet no evidence as to whether most or all ofthese diverse lateralisation phenomena are expressions ofone system of vertebrate-conserved left-right information inforebrain development, or are homologous in any real waywith the system that ultimately underlies hominoid hand-use/linguistic partitioning. But it can be seen how in ageneral way, complementary lateralisations of brain func-tion are advantageous to complex processing. The subpro-cesses of cognition, motor organisation or decision-taking,though ultimately needing to be brought together to result ineffective behaviour, can each largely occur within onecoherent neural subsystem or network, thus minimisingthe need for continual crossing of midline commissures by

half-processed information. This may be especially so in thenon-mammalian vertebrate where the relative ‘channelcapacity ’ for left-right cross-transfer is less than that offeredby the corpus callosum.

It has perhaps been a counter-intuitive finding, thathuman functional cerebral lateralisation appears indepen-dent of visceral situs in its mechanism. Because of tantalisinglack of information, less can be said developmentally aboutthese other vertebrate neural lateralisations, though whatwe do know appears currently confusing. Andrew (2000;see also Andrew et al., 2000) proposes a daring, deep-evolutionary hypothesis as to their origin which relates tothe hypothesis of a non-symmetrical chordate ancestordiscussed in Section III, and the progressive change oflifestyle accompanying vertebrate origins. While that pro-posal may seem imaginative to the point of fantasy, it shouldbe recalled that the idea of non-symmetry (dexiothetism)within vertebrate ancestry has itself been treated withextreme scepticism, whereas a balanced assessment of allthe sources of evidence strongly indicates that it should beconsidered seriously. Andrew’s (2000) hypothesis wouldsuggest that the neural functional lateralisations he considersare deeply linked to situs developmentally, and would thusbe expected to share its extreme directional reliability. Evenhad the intuitive expectation proved correct that humanhand-use/linguistic lateralisation shared developmentalcues with situs, the presumption would have had to bethat this evolutionarily recent phenomenon had ‘ tied’ itsdirectionality to situs via a weak linking mechanism to ac-count for the reduced ‘reliability ’ that is observed. But anyneural lateralisations that co-evolved with situs itself, startingfrom a post-dexiothetic animal in which left-right re-symmetrisation through sense-organ pairing etc. had noteven begun (see Section III.2), should be genetically integralwith situs development.

Mammal embryo torsion in utero is only likely to be thesource of at most very weakly and unreliably directionalstimuli for neural lateralisation. Rogers (1990) on the otherhand, has good evidence that the torsion of the chickembryo within the egg-shell, by selectively occluding one eyefrom light stimuli during a particular late pre-hatching time-window, is responsible for the particular lateralisations offorebrain functions she studies. This would indeed makethese lateralisations a consequence of the situs mechanism innormal development, since the latter controls the constantdirection of axial torsion. This particular lateralisation mightbe expected substantially to share in the directionalreliability of situs, with spontaneous reversals being verymuch rarer than those of human left-right neural function,although the dependency would be very indirect rather thanthrough situs gene cascades as such acting in the brain.Reverse-torted bird embryos developing in ovo are rare (lessthan 1 in 103). The puzzle is, that while the particular left-right partitioning in precocial bird behaviour studied byRogers (1990) may indeed be very reliable under normalconditions, many of the other phenomena that she andothers have reviewed show much less reliable directionalitythan this.

A final piece of information concerns the diencephalicallylocated asymmetry, particularly prominent in many fish,

398 Jonathan Cooke

reptile and amphibian species, whereby the habenularganglia forming from the right and left dorsal neural tubeare differently sized, along with asymmetry of the ac-companying parapineal body. Though its anatomical ex-pression may differ strikingly in form in different species,the asymmetry as such is clearly a conserved feature andpresumably has some present or historical significance. Ithas long been known to be aligned with visceral situs in beingpreferentially disturbed or reversed in the right-handmembers of experimentally produced conjoined twins (seeSection III.2). It has thus been an exciting finding that adistinctive phase of left-sided expression of the phylotypicsitus gene cascade of nodal, lefty and Pitx homologues occurswithin zebrafish diencephalon (Concha et al., 2000; Lianget al., 2000). This normal expression is necessary for thedirectionality of the habenula/parapineal asymmetry in thatspecies, though not the development of the asymmetry per se.An earlier non-neural, non-lateralised phase of nodalsignalling first represses any diencephalic expression of thenodal cascade. Later experimental rescue of nodal functionin mutant embryos allows their development to hatchingand maturity, but with either absence or bilaterality of thediencephalic situs cascade. In such fish the situs-relatedasymmetries develop with random directionality, althoughsome lateralities, such as those of heart and this diencephalicanatomical one, are correlated in individuals.

Behavioural lateralisations involving differential eye usein approach and agonistic behaviours are already charac-terised in normal zebrafish (Miklosi & Andrew, 1999;Andrew, 2000). Such lateralisations can now be examinedin these experimental fish, but interpretation of the resultsmay be limited to correlating abnormal situs, rather than thespecific diencephalic gene expressions mentioned, withabnormality for the behavioural lateralisation concerned.The reason for this is that it is not known which, if any,lateralised behavioural functions are specifically subservedby this habenular/parapineal asymmetry. Equally likelya priori, is the possibility that lateralisation of other forebrainneural subsystems, not obviously connected with this one,nevertheless lie downstream of these phylotypic left-sidedgene expressions in fish forebrain. A positive finding ofdisturbed behavioural lateralisation in this work would behighly noteworthy however, in that it would contrast withthe apparent independence of human ‘hand-use/linguistic ’laterality from situs, and lend credibility to ‘deep-phylogen-etic ’ conjectures about the ultimate origin of at least someneural functional lateralisations.

V. IMPLICATIONS FOR DIRECTIONAL

ASYMMETRIES IN PROTOSTOME ANIMALS

A view of the origins of vertebrate left-right structure such asthat advocated here has implications for our understandingof the sporadic occurrences of directional (handed) anatom-ical asymmetries elsewhere in the animal kingdom. Thewell-known dextral versus sinistral coiling of the structures ofgastropod mollusc species, susceptible to genetic reversal ona simple and maternally inherited basis, can be set aside

here as derived from the tilting of the planes in thedeterministic spiral cleavage pattern of these organisms(Verdonk, van den Biggelaar & Tompa, 1983). In nema-tode development, left-right asymmetrical structure withthe characteristic cell lineages that lead to it can likewisebe reversed by mechanically reversing early asymmetricalpositioning of cleavage products (Wood, 1991). In that thecell division apparatus utilises chiral (microtubule-based)molecular assemblies, these instances of global developmen-tal asymmetry are likely to be derived rather directly fromsuch molecular cues.

Normal directionalities of the spiral texture of thearthopod gut, as well as the ability of certain mutations tocause a spiral type of ‘ slippage’ in the external cuticularpattern in Drosophila melanogaster (Martin-Blanco & Garcia-Bellido, 1996), most probably derive from the expression ofchiral molecular structure within connective tissue at asupracellular level, rather than from global gene-expression-encoded information as to right and left identity of tissues.Asymmetries of the structure of insect genitalia, derivedfrom the products of bilateral pairs of multicellular genitaldiscs, are perhaps explicable on a similar local torsionalbasis, ultimately capitalising on mechanical structure intissue. It is doubtful however, that protostome animals havesystematic developmental access to global left-right infor-mation, encoded as gene expressions that mark left versusright identities of rudiments within the bilateral body plansuch as we have seen to underlie vertebrate situs. Thus, whileselection regimes on Drosophila populations can quite easilyincrease, or even marginally decrease, normal levels offluctuating asymmetry between bristle patterns on the twosides of individual flies, they have been unable to imposedirectional asymmetry on such patterns (Maynard Smith &Sondhi, 1960; Coyne, 1987; Tuinstra, DeJong & Scharloo,1990). In the apt words of Tuinstra et al. (1990), ‘Left andright are not distinguished in development ’.

A few species of decapod crustacean, sporadically dis-tributed taxonomically, exhibit strong population direction-ality for the size difference between right and left cheliceraethat is typical of these forms (reviewed in Palmer, 1996).This can occur even without the obvious ‘environmental ’cue that is provided to hermit-crabs ! Since these append-ages develop from nests of cells relatively far from themidline, such directionality would seem to require morethan stably propagated molecular chiral or cleavage planeinformation. The taxonomically sporadic occurrence ofsuch directionality within arthropods however, togetherwith the organisational advantages to be derived fromsystematically left- and right-specifying gene expressionsthat this article has suggested for vertebrates, indicates thatsuch gene expressions are not and cannot be developedwithin protostome phyla.

Most recently and intriguingly, a small but consistentdirectional asymmetry in wing size and shape, conserved indirection and character among drosophilid and muscid flyspecies, has been described (Klingenberg, McIntyre &Zaklan, 1998). This has been adduced as counter-evidenceto the assertion that such animals lack any ‘ left-right axis ’ inthe sense the term has been applied to vertebrates. I suggestthat the clearly gratuitous nature of this particular left-right


difference indicates that rather than having positivelyevolved, it is in some sense ‘necessary’, being the result ofa limitation on the achievable accuracy of mirror-imaging,within the mechanism of insect wing morphogenesis, due toinherently chiral components in the biological buildingmaterials. It is hard to imagine why a directional size-shapedifference between the wings of a flying insect should haveevolved, as opposed to having been tolerated by evolution,particularly as individual flies fold their wings with randomupper/lower positioning on alighting (M. Averoff, personalcommunication). What could be the nature of such adirectional limitation on the accuracy of mirror-imaging inshape formation? The cytoskeleton becomes organised ona cell-wide basis, particularly in flattening epithelial cells.Under the extreme conditions attending wing-blade for-mation a small but appreciable bias, or constraint onmorphogenetic replicability, might be imposed by the factthat on either side of the midline, the overall shape beingproduced bears an opposite relationship to the intrinsiccytoskeletal chirality within the participating cells. It couldbe that fly wings are in fact as near mirror-symmetrical, inoverall size/shape, as selection can make them with thebiological building blocks available.

This in turn raises the possibility that subtle left-rightdifferences of mirror-image shapes in multicellular structure,initially present as the result of inherent limitations imposedby chiral molecular assemblies, might subsequently becapitalised upon when a selective force for development ofa directional asymmetry arises in this part of the animalkingdom. In this way, for instance, occasionally in particularspecies, the developmental conditionality that crab chelicer-ae should initiate positively different growth regimes be-cause they formed respectively on the animal’s right and leftsides might be achieved, without any crab tissue ever beingencoded with gene expressions for ‘right ’ or ‘ left ’ characterper se as occurs in a vertebrate embryo. This necessarilymuch more limited basis for development of left-rightdirectionality, leading to sporadic examples only of suchdirectionality, may characterise those protostome embryotypes that do not establish massive asymmetry that is directlypropagated from an initial ‘ spiral ’ cleavage pattern, as ingastropod molluscs.

A somewhat bold (and thus fragile) hypothesis mighttherefore be that, apart from recourse to ‘memory’ derivingfrom spiral or other asymmetric cleavage planes, or anability to utilise chiral molecular architecture locally,protostome bilaterians generally have had no means ofevolving any widespread encoding of left and right origins intheir tissues. Such coding has been conferred on deuter-ostome body plans only, because of the contingent dexio-thetic transformation that lies within their shared ancestry.An alternative scenario would of course be that a geneticlinkage using the opposing relations between chiral micro-structure and macro-anatomical structure, across a midline,is primary to all directional asymmetries between right andleft paired organ rudiments in bilaterian animals. Instead ofdexiothetism, chordate-like forms might just share theinnovation that this linkage has been elaborated into theleft and right gene cascades. But this again begs the questionas to why, since a capacity for such lateralised gene marking

is so advantageous, it has not arisen at any other deepposition within metazoan phylogeny. It also fails to accountfor the ‘gratuitous ’ nature of some prominent anatomicaland gene expression asymmetries in protochordate bodies.

The difference in inherent left-right organisational po-tential between protostome and deuterostome embryos thatI propose may have constituted a non-trivial, historicallycontingent differential ‘developmental constraint ’ (MaynardSmith et al., 1985; Raff, 1996) ; one that has been ofconsequence indeed in evolution. Given the advantages tocomplex neural processing that have been proposed fordirectional functional complementarity in brain halves, itwould be fascinating to search for directional lateralisationin say the bee’s brain, or that of the octopus (a molluscdeveloping without benefit of any stereotypical cleavageplan). In these structures, such lateralisation should surely befound if intrinsically attainable ! If indeed it were found, itwould be especially interesting to see whether lateralisedgene expressions are developmental causes rather thanconsequences of the lateralisations.

VI. CONCLUSIONS

(1) Situs is a vertebrate-conserved left-right organisationprincipally expressed as a co-ordinated packing arrange-ment of the gut, vascular system and (where relevant) lungs,but extending in more subtle ways into the axial body wall(embryo torsion and perhaps left-right somite segmentstaggering), and into some subset of neural asymmetries thatincludes petalia (the slight differential rostro-caudal position-ing of left and right cerebral cortices in many primates)and the habenular/parapineal asymmetry. However, atleast one neural functonal left-right asymmetry apparentlydevelops quite independently of situs. This is the one which,while its precursors may lie quite deep within vertebratephylogeny, ultimately underlies human language capacityand skill lateralisation.

(2) Despite their independence at the population leveland within individuals, both situs and the human hand-use/linguistic neural lateralisations are liable to perturbationsassociated with the circumstance of ‘ late twinning’, wherethe two body patterns of monovular twins have begun to bespecified before the loss of cellular continuity in embryonictissue. This can be formally understood in terms of the ex-posure of one of the two parallel axial patterns to abnormalarrays of lateralising signals, due to presence of the otherone, even if the signals concerned are quite different forthe situs-mediating and the neural functional asymmetrysystems.

(3) It is clear that a core universal ‘phylotypic ’ cascade ofleft-specifying gene expressions (and probably of opposing‘right ’ ones) accompanies the most closely anatomicallyconserved or ‘pharyngula ’ stage of vertebrate embryo-genesis. Certain genes less central to these cascades mayhave ‘swapped’ their sidedness of function during vertebratediversification, though multiple successive gene roles andother interpretation challenges make this difficult to assert.If the pre-gastrular vertebrate embryo goes through a truly

400 Jonathan Cooke

bilaterally symmetrical stage, then chiral (i.e. ‘handed’)molecular structure, aligned among the cells, must surelyprovide the basis for mechanisms of directional symmetry-breaking and correct initiation of the cascades. While uni-versality is currently less clear for this initiating mechanism,there are striking indications that at molecular depth it mayalso turn out to be conserved, or at least drawn from amonga small universally available set of mechanisms in all ver-tebrates. Evidence concerning a transversely orientated ex-tracellular micro-flow due to ciliary action, that is presum-ably a reflection of chiral macromolecular assembly andaligned anchoring within cells, has recently strengthened formouse. But this may operate at different stages, or not at all,in embryos representative of other vertebrate classes. Thereis considerable evidence for the primacy of vectorial trans-port mechanisms within the joint intracellular space, via gapjunctions, in vertebrate developmental symmetry-breaking,and these may begin operation at very early stages.

(4) There is a plausible evolutionary ‘ just-so-story’ as towhy situs should represent an optimal arrangement interms of the efficiency and reliability of vertebrate adultfunction, although like most such hypotheses, this is un-testable. A dominant community assumption, among thoseresearching the mechanisms, currently appears to be thatsymmetry-breaking and laterality-propagating gene cas-cades evolved de novo with the origin of the vertebrate or thechordate clade, in response to this adaptive ‘desirability ’.This implies the belief that the ur-vertebrate or ur-chordatewas an untransformedly bilaterian animal, in an equivalentsense to that in which, say, an insect or a polychaeteworm is bilaterian (albeit with somehow inverted dorso-ventral organisations – see De Robertis & Sasai, 1996;Gerhart, 2000).

(5) A different view, while accepting the compellingmolecular evidence that deuterostome animals share anoverall ancestor with other groups of phyla to constitute theclade Bilateria, would nevertheless propose that most or alldeuterostomes, certainly including chordates, share in theirancestry a subsequent transformation in which the primor-dial dorsal-ventral dimension became a left-right one. Thecentral consequence of this idea would be that within thenewly axial streamlined body, that developed aroundthe time of vertebrate origins, a profound developmentalgenetic organisation would already have been in place torespond to any selective pressure for the further elaborationof reliably directional visceral left-right asymmetries. Therewould have been no need for de novo invention. This left-right organisation within the internal ‘visceral ’ vertebrate orchordate (the ancestral ‘visceral ’ body: Romer, 1972) wouldcorrespond, in the simplest interpretation, with an originalventro-dorsal organisation.

Various detailed candidate scenarios for the derivation ofvertebrate form from non-symmetrical echinoderm-likeones, based on fossil interpretation (cited in Section III.2),are fascinating for the appropriate specialists. For mostcellular and molecular biologists, however, persuasion as tothe overall plausibility of proposed scenarios in vertebrateancestry cannot come primarily from structural interpret-ations of fossil forms that have no close living equivalents.They must, rather, consider the combined evidences from

comparative anatomy and embryology of extant deuter-ostome forms, from molecular phylogeny, and most strik-ingly, from comparative developmental expression patternsof gene orthologues. I find this combined evidence to be quitecompellingly in favour of the hypothesis that current ver-tebrate left-right organisation derives from the co-optation ofan ancient dimension of organisation in a non-symmetricaldeuterostome ancestor. The most highly elaboratedscenarios based on interpretations of fossil traces propose a‘dexiothetic ’ transformation in which an original right-handside became a new ventral or ‘ lower’ surface, at least in theadult form. This form, probably a sessile or slow-movingbenthic filter-feeder, was essentially non-symmetricalthough it may have been axial. The subsequent history ofarriving at the vertebrate degree of external bilateral sym-metry has been one of sequential re-acquisition of new‘right-hand’ structure to symmetrise the feeding apparatusand the nervous system, perhaps with re-rotation, sym-metrisation and forward invasion of the locomotory ‘ tail ’. Aconverse ‘ laevothetic ’ interpretation has been proposed,perhaps motivated partly by the need to emphasis the fra-gility of fossil interpretation in deep evolutionary time.Nevertheless, either scenario seems more consistent with theevidence of all types, taken together, than does one wherevertebrate ancestry lacks such a transition from primary(‘ur ’) bilaterality.

(6) In relation to this overall interpretation of the originsof vertebrate left-right, the question arises : why is thereapparently a contemporary requirement for molecularmechanisms that break cellular symmetry in the embryowith each ontogeny? One possible explanation is that the re-symmetrised lomotory/neural component of vertebrateanatomy has become so pervasive as to reach back into theearliest embryo stages, obliterating those cues that allowedsetting up of the original bilaterian dorso-ventral – morerecently a right-to-left – organisation. To preserve thefunctionality of that axis a new mechanism could be re-quired, relying on molecular chirality within cellular struc-ture as the only possible cue, that triggers the still extantregionalising gene expressions with the correct direction-ality. It seems more likely however that throughout deuter-ostome history the larval form has always retained truebilaterian organisation, and that since a dexiothetic orequivalent transition, production of adult forms in a meta-morphosis or ‘settling ’ event has involved a mechanismensuring a consistent derivation of ‘ lower’ substrate-appliedversus ‘upper ’ surfaces (expressed in extant echinoderms as‘aboral-oral ’) from this larval structure.

Among extant forms of development, the settling of acrinoid echinoderm larva in which the left gastrular side cor-responds with the upper ‘oral ’ adult one, may most closelyembody the original form of this ontogenetic transition.The reliable directionality of echinoderm larval settling isnot, to this author’s knowledge, understood mechanistically,but given the small scale of structure involved and theprominent use of cilia in larval locomotion, it may not be toofanciful to ask whether the chiral nature of ciliary structureand action is, or has been, involved here as well. Whether ornot that detailed speculation is correct, the ‘symmetry-breaking’ event in extant vertebrate development may best


be understood as, in some sense, a recapitulation of anancestral mechanism while the contemporary embryo isat a gastrula stage of organisation corresponding with thatof the ancestral larva. Alternatively, the developments ofurochordates, acraniates (Amphioxus etc.) and vertebratesmay be linked within the deuterostomes by having jettisonedthe primordial larval form. The developing acraniate,urochordate and vertebrate are not ‘ larval ’ in the same senseas the ciliated planktonic echinoderm and hemichordatestages. In this regard it is noteworthy that, while Amphioxushas not yet been appropriately investigated, evidence forleft-right organisation at the earliest cleavage stages has nowbeen found in embryos of the other two groups.

(7) The concept of recapitulation seems to have a badpress among contemporary evolutionists, but despite theprofound plasticity of form shown by early development,arbitrary characters that almost compel recapitulationaryexplanation surely do exist for ‘postlarval ’ phases of chor-date development. The striking, transient gastrular struc-tural asymmetry shared by at least some birds (Section III.2.)is one such character. The developmental progression ofhead and branchial anatomy in Amphioxus, and the ‘ver-tebrate phylotypic ’ left gene-expressions that it shares withthe developing tunicate, may be others.

VII. ACKNOWLEDGEMENTS

I thank John Maynard Smith, Adam Wilkins, Michael Akam,Thurston Lacalli, Nick Holland and anonymous reviewers for dis-cussions, insightful and scholarly comments on drafts, and helpwith hard-to-access literature during the preparaton of this review.I am particularly grateful to Michael Akam and colleagues in theZoology Department at Cambridge for enjoyable and stimulatinghospitality during the period of its incubation.

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developmental mechanism and evolutionary origin of vertebrate left/right asymmetries

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