The evolutionary origins andsignificance of vertebrateleft–right organisationJonathan Cooke
SummaryIn the last few years, an understanding has emerged ofthe developmental mechanism for the consistent internalleft–right structure, termed situs, that characterisesvertebrate anatomy. This involves largely vertebrate-conserved (i.e. ‘phylotypic’) gene expression cascadesthat encode ‘leftness’ and ‘rightness’ in appropriatetissues either side of the embryo’s midline soon aftergastrulation. Recent evidence indicates that the initial,directional symmetry breaking that initiates these cas-cades utilises mechanisms that are conserved or at leastclosely related in different vertebrate types. I describe ascenario whereby the capacity for directional modi-fication of an otherwise bilateral body plan can be viewedas an adaptive innovation rather closely connected withvertebrate origins, enabling optimal ‘design’ for veryactive lifestyles. But an alternative scenario, while re-taining the view that situs and indeed other vertebratefunctional lateralisations are deeply adaptive, pro-poses that they originated in the co-optation of left–rightdevelopmental information inherited from a very earlystage in metazoan diversification. It is proposed thata remote chordate ancestor lost its original or ‘ur-bilaterian’ symmetry to pass through an altogether non-symmetrical stage, and that the vertebrate dorsoventralmidline plane is not descended from that original one.I review the considerable evidence in favour of thisscenario, and discuss its wider implications for direc-tional asymmetries across the Metazoa. BioEssays26:413–421, 2004. � 2004 Wiley Periodicals, Inc.
Outline; levels of interest in study of
vertebrate left–right organisation
Most of us are aware that our outwardly bilaterally symmetrical
form hides a systematic left–right disposition or ‘packing’ of
our major visceral organs, coordinated among subsystems
suchasheart,major bloodvessels, gut andassociated glands.
The directionality of this asymmetry, known as situs, is clearly
conserved among our vertebrate relatives from a latest com-
mon ancestor, despite their varying degrees of elaboration of
visceral structure. Coordinated directionality as between the
major organ subsystems is clearly functionally important,
since instances of human developmental situs incoordination
(order of one per 103 births) show variably but often severely
reduced viability. But fascinatingly, the direction of the whole
asymmetry when coordinated appears to be of no functional
relevance. Much rarer (1 in 104) individuals have internal
anatomy that is the complete mirror-image of normal, yet their
incidence was not ascertainable until recently since most of
them live normal lives unaware of their singularity.
It would not have been a safe a priori assumption that
a macroscopically left–right reversed vertebrate anatomy
would function every bit as well as the normal. At the level of
cell structure, biology is intrinsically ‘handed’ i.e chiral. In
particular, various key protein assemblies such as cilia and
their basal bodies cannot be aligned so as to have equivalent
right and left sides, if they are orientated or anchored in the
cells of an embryo with respect to its dorsoventral or antero-
posterior axes. One or more aspects of bodily function might
thus have been compromised and inefficient unless gross-
anatomical and cell-structural ‘handedness’ were in the parti-
cular relation that had evolved as ‘normal’. While, in fact, this
relationship turns out not to matter at all for normal vertebrate
function, chiral molecular activity is somehow undoubtedly
central to normally directional symmetry breaking in the
embryo for gross anatomical pattern.(1) This provides a strong
explanation for the evolutionary, as well as within-species con-
servation of normal situs direction.
Vertebrate development involves the formation of various,
frequently paired, organ rudiments across considerable tissue
distances and over an appreciable time period. Subsequently,
many of these undergo left–right differential morphogenesis,
growing in coordination with each other to give the packing
arrangement seen.Amechanismmaximising reliability of situs
development might thus incorporate one initial ‘symmetry-
breaking’ signal at an early stage, triggering distinctive,
mutually exclusive left and right cascades of gene expression
that propagate through development. Then at any subsequent
time and anatomical location, tissue in at least one germ-layer
of the embryo would be labelled with its lateralised identity
before beginning its contribution to morphogenesis, thus
coordinating the directionality with which the individual organ
rudiments develop their left–right structure. This turns out to
BioEssays 26:413–421, � 2004 Wiley Periodicals, Inc. BioEssays 26.4 413
Museum of Zoology, The University of Cambridge, Cambridge, UK.
E-mail: [email protected].
DOI 10.1002/bies.20015
Published online in Wiley InterScience (www.interscience.wiley.com).
Hypothesis
be indeed how situs is controlled developmentally. A variety
of pathological anatomies (heterotaxias—internal left–right
confusions, or isomerisms—partial or complete double-‘right’
or double-‘left’ structure) occur when the symmetry-breaking
signal has failed, or the right or left tissue-labelling cascade
has later failed at particular times or locations. Only much
more rarely does symmetry breaking fail or get subverted at
the earliest time, leading to entire, coordinate reversal, situs
inversus.
One might conclude that, viewed as a vertebrate evolu-
tionary invention (or a chordate one—see later), situs ‘could
have turnedout’ to showeither directionality, or indeed random
directionality, provided only that a mechanism ensured its
coherence within each individual. In human terms, the latter
could havemeant that left and right were nomore than apurely
abstract social convention—perhaps arising recently from the
need to communicate about human navigation, for instance.
It is a fascinating realisation that we owe to the robust, chiral-
molecule-driven mechanism of our developmental symmetry-
breaker, the fact that these termsmeanmuchmore than that to
our societies.(2)
Below, I outline the developmental arrangements whereby
right and left cascades of tissue-labelling gene expression are
stabilised in order to show that, in fact, these form a typical
example of an embryo axial patterning system. Then, I explore
a tension that often accompanies ‘explanation’ of biological
organisation. This tension is between, on one hand, the belief
that, mechanisms simulating optimal design can always arise
de novo in response to new selective pressures on function
and, on the other hand, the idea that historical contingency
constrains and shapes evolutionary opportunities. I outline a
purely adaptationist scenario, according to which situs could
have evolved, in a fundamentally symmetrical vertebrate-like
ancestor, as an optimal engineering response to demands of
a newly active, predatory lifestyle. However, I then review the
evidence indicating that, even while adaptive, the vertebrate
mode of encoding right and left identities within tissues
represents a co-optation of a deep ancestral feature of the
clade of animals to which we belong. This feature, not shared
with other lineages of bilaterian metazoans, was a morpholo-
gical transformation whereby our ancestors had become
fundamentally non-symmetrical as adults.
The molecular developmental biology
of vertebrate situs: loose ends
Many excellent, detailed reviews of the vertebrate left–right
molecular-geneticmechanismarenowavailable (for example,
Refs. 3–6), and the list of genes and signal pathways involved
continues to expand, some possibly acting in parallel rather
than in one cascade. There is nevertheless vertebrate con-
servation of particular ‘phylotypic’ gene expressions, certainly
for left and probably for right identity, centred in mesoderms
lateral to the gastrular midline and commencing some hours
into gastrulation. At this stage, axial midline structure is already
present to divide the embryo, and particular gene products
activated in this midline additionally serve to block inappropri-
ate cross-invasion of the ‘left-phylotypic’ intercellular signal,
the nodal protein. A further, earlier-acting set of right and left
intercellular signals, more locally expressed and near the
midline, have to date been studied only in bird (chick and quail)
embryos. These are not evident in either the yolky embryos of
zebrafish and frog, or the highly morphologically ‘derived’
mouse version of vertebrate development. In such embryos,
nodal seems to be the first easily recognisable lateralisedgene
expression,(7) whereas the earlier cascade in birds precedes
and seems to trigger the left nodal expression leading into the
phylotypic postgastrular sector.(8–10) The possibility remains,
however, that this difference of organisation tracks with
gastrular morphology rather than truly with phylogeny. Thus
the ‘blastoderm’—type rabbit embryo resembles chick more
than mouse anatomically, and perhaps in its left–right gene
expressions, with deployment of Sonic hedgehog (Shh) and
FGF-8 signal geneexpressions to left and right at its equivalent
of the avian Hensen’s node.(11)
Left-lateral expression of the homeobox-containing Pitx-2
transcription factor, once triggered by the nodal signalling
pathway, exhibits developmentally prolonged expression
consistent with a role in maintenance of ‘left’ character in
morphogenesis for a variety of organ primordia (viscera, lungs
etc.) as well as for left-specific cardiac wall structure. A
reciprocal right-sided role, but one more restricted in space
and time, may be played by the zinc finger-containing trans-
cription factor Snr (related to the Drosophila Snail gene;
sometimes known just as ‘vertebrate snail ’). This appears to
control the direction of heart looping and of embryo torsion, but
also to participate in the transmission of ‘rightness’ by initially
confining ‘leftness’; i.e.Pitx-2 expression, to its proper side.(12)
SnR itself is repressed by nodal signal. Indeed, the nexus of
positive feed-forward and negative feed-back regulatory
relationships among the ‘left–right’ gene expressions, ensur-
ing their appropriate spatial exclusivity and temporal ordering,
is strongly reminiscent of other primary pattern-forming de-
velopmental gene cascades that are understood, e.g. the
Drosophila gap-to-segmentation or the vertebrate gastrular
dorsoventral systems. A forthcoming review(13) deals with
these aspects among others.
It has long been realised that, if the early embryo is
genuinely bilaterally symmetrical in cellular anatomy, then
reliably directional breaking of that initial symmetry can only
derive, ultimately, from the orientated chiral structure of one
or more macromolecular assemblies within its cells.(14–16) If
somehow tethered and alignedwith one ormore other embryo
axes (anteroposterior, or apicobasal within an epithelium),
such molecular assemblies could initiate handed asymmetry
across a midline, for instance by a net left–right component of
intracellular or intercellular transport of an initiating signal
Hypothesis
414 BioEssays 26.4
molecule. Cilia and their basal bodies had attracted specula-
tion because of their obviously chiral or ‘handed’ structure and
mechanical activity (a spiral component to the beat), and their
defined mode of tethering in cells. It has now been reported
that the normal beating of a densely distributed group of
atypically structured cilia (‘protocilia’) produces a flow to the
mouse embryo’s left within the medium bathing the archen-
teric epithelial surface, and that this flow is necessary and
determinative for normal directionality of the lateralised nodal
and Pitx-2 expressions that stabilise situs development.(17,18)
The authors propose that, at least in mouse, developmental
symmetry is broken by ciliary driven leftward extracellular
flow of a ‘morphogen’, possibly nodal protein itself or a related
protein, whereby phylotypic left nodal gene expression is
activated in the gastrula.
This discovery has seemed to rationalise a diversity of data
from knockout mice, in which absence of gene functions
associated with the building or activity of cilia correlates with
heterotaxia or with random ‘flipping’ to situs inversus, as well
as a human familial syndrome inwhichmalfunction of all cilia in
the body correlates with random situs direction.(14) Ciliary
structures that are clear homologues in molecular terms
with the mouse nodal protocilia are observable in several
vertebrate embryo types, at times just preceding their first
‘phylotypic’ left nodal expressions.(19) Nevertheless, as
recently reviewed,(1) extensive data now suggest that these
observations may not have defined the truly initiating, con-
served vertebrate symmetry-breaking mechanism. Left–right
directional net transport processes get underway within the
joint intracellular space, considerably before gastrulation
begins. The data hint at a plurality of other (pleiotropic) roles
for gene products involved in building cilia/basal bodies, and
such roles also provide opportunity for the derivation of left–
right directionality from the chiral structure of an anchored
molecule through intracellular transport processes. In parti-
cular, the community awaits elucidation of the function of the
intracellular protein Inversin, the remarkable inv gene product
whose malfunction reverses, rather than randomising situs,
and which is now reported to be associated with protocilia.(20)
inv mutant mouse embryos are reported to show detectably
abnormal, but clearly not reversed nodal ciliary flow!(21)
Evolutionary origins of vertebrate left–right;
optimal design versus historical constraint
and co-optation
Most ‘major body plan’ features are less transparently
adaptive in origin than are, say, the design features of wings
or skin, though such lack of present transparency means little.
But among body plans, the vertebrate one, including situs,
perhaps has some of the best adaptive credentials. Onset of
an active searching lifestyle, probably accompanied by pro-
gressive size increase, probably marked vertebrate origins.
In addition to the extra demands for metabolic rate accom-
panying such a lifestyle change, the ‘square-cube’ law dictates
that, for bodies of identical form, totalmetabolic demandwould
increase more rapidly with linear size than would available
internal area for absorptive and exchange processes. There
would thus have been a selective premium on relative in-
crease in the length and complexity of formerly simple tubular
viscera and circulatory vessels, within a symmetrically stream-
lined outer ‘locomotory’ body wall. For processes like nutritive
absorption and circulatory metabolite exchange, the relation-
ship is intuitively clear, but recent analysis of vertebrate heart
function, for instance, has demonstrated strikingly how, for a
given mass of muscle, the spirally coiled form has much
greater pumping efficacy than would a simple valved tube.(22)
Development guided by body-wide cascades of left- and right-
specific gene expression has obvious advantages, ensuring
that near-identical packing arrangements among the complex
viscera can occur in every ‘normal’ individual, rather than a
random series of mostly suboptimal ones. Complexity in the
organ systems can then further co-evolve. Such intimate,
cooperative fine tuning between separate organ systems is
manifest when the first symptom of acute human heart
malfunction resembles indigestion—or when acute indiges-
tion forces arrhythmias in the otherwise healthy heart!
Incontestably, in view of molecular phylogenetic data, ver-
tebrates belong to the great animal clade known as Bilateria.
If the assumption is made that the proto-vertebrate had
descended from its ur-bilaterian ancestors with the original
dorsoventral plane of bilateral symmetry intact, directional
left–right organisation could then only occur by de novo
‘invention’ of a symmetry-breaking mechanism. One form of
this scenario, namely that the whole situs mechanism
including the derivation of symmetry breaking from molecular
chirality was a vertebrate adaptive innovation, has often
been assumed by molecularly trained workers who lack
acquaintance with the fields of comparative anatomy and
phylogenetics.
Recent comparisons of developmental expression pat-
terns for nodal and Pitx orthologues in the protochordates
Amphioxus (Cephalochordata) andCiona (Urochordata) have
already forced a revision of this view. There are clear and
striking left-lateral expressions of these genes during larval
stages most equivalent to the vertebrate neurula/pharyngula.
At the least, the asymmetry innovationmust haveoccurred in a
more remote chordate-like ancestor rather than just a proto-
vertebrate (reviewed in 23). But even this scenario runs into
trouble when a wider range of data is considered. The striking
developmental asymmetries of anatomy in the head and gill
region of Amphioxus (reviewed in 24), and of gene expression
even without associated anatomical features (also in Am-
phioxus but especially in the urochordate tadpole larva(23)),
themselves seem functionally gratuitous. Nothing currently
indicates that these forms had ancestors that led active
vertebrate-like lives, with the functional demands for organ
Hypothesis
BioEssays 26.4 415
packing that the adaptationist scenario for the origin of situs-
like asymmetry requires (though such arguments are inher-
ently insecure). Amphioxus is small and inactive enough to
need no functional heart.
Amphioxus shares with adult urochordates a simple direc-
tional looping and asymmetrical structure of the viscera that
could plausibly be homologous with that in extant echino-
derms. The simple gut looping in extant echinoderm classes
bears a conserved directionality in relation to the oral–aboral
axis, within their pentaradially symmetrical adult body plan.
Echinoderms, hemichordates (acorn worms) and the above-
mentioned ‘protochordate’ types along with vertebrates are
now accepted on molecular phylogenetic grounds as repre-
senting a clade, the Deuterostomia.(25) Comparative anato-
mists recognise a set of structural features that links these
diverse forms (radial cleavage, lack of correspondence be-
tween mouth and blastopore at gastrulation, larval forms,
notochord, pharyngeal gill slits), even though each group only
manifests a subset of them. As bilaterians, deuterostomes
share an ancestry with all those other animal groups in which
the Hox/Hom cluster of homeobox genes is deployed as the
basis for a longitudinal (anteroposterior) axial organisation.
But earlywithin their ownphylogeny there occurred, either as a
single founding transformation or as a repeating tendency to
such transformation, a radical departure from ur-bilaterian
organisation in adult forms. In extant echinoderms, a sub-
stantial trace of properly bilaterian, co-linear deployment for
Hox orthologues occurs along the bilateral coelomic pouches
of the larva.(26) But in the internal reorganisation of metamor-
phosis, the adult ‘oral–aboral’ axis arises and is not aligned
with either the larval dorsoventral or longitudinal ones. The
remaining deuterostome forms show, to varying extents, the
appearance of bilaterality in parts of their bodies coupled
with (gratuitous-seeming or functional) left–right structure in
others.
We must then ask, does being bilaterian by descent
necessitate that left–right organisation, when it occurs within
deuterostome bodies, directly corresponds to ‘ur-bilaterian’
left and right? The ancestral ur-bilaterian organism presum-
ably had a dorsoventral cross-sectional organisation, which
with its anteroposterior axis defined right and left sides. But
this would not have provided a molecular basis for encoding
its mirror-image sides with ‘leftness’ and ‘rightness’ as such.
An alternative idea is that, during bilaterian diversification, a
transformation occurredwithin the deuterostome lineage.One
original ‘ur-bilaterian’ side progressively became a new
‘ventral’ side and the other, the new ‘dorsal’ side, in essence,
a 908 rotation.(24,27,28) This would most probably have oc-
curred to give rise to a less axially organised, slow-moving
or sessile, detritus- or filter-feeding adult. But, in reponse to
evolving lifestyle, descendants of such an organism might
reacquire to varying extents a symmetrical bilateral outer
structure. They might nevertheless retain the developmental
organisation of what had once been a dorsoventral dimension,
now co-opted as the capacity to encode genetically regiona-
lised and thus independently evolvable right and left internal
structure. At this point, it should be mentioned that there
are plausible alternative explanations for the appearance of a
literal 1808dorsoventral inversion having occurred as betweenprotostome and deuterostome (arthropod versus vertebrate)
body plans.(29–31)
The above type of 908 transformation was originally pro-
posed to have occurred after the origin of hemichordates,
making the latter the only deuterostomes retaining ‘ur-
bilaterality’. But molecular phylogeny now suggests that they
and the (as adult) radially non-bilateral echinoderms are sister
groups, and together form a sister group to all other, more
chordate-like forms.(25) Thus the transition might be a deuter-
ostome-defining character, with hemichordates the group that
has most profoundly reacquired symmetry. The adults of just
some hemichordate species retain a feature shared with
echinodermmetamorphosing larvae, which pre-figure the adult
axial transformation in that only the left protocoel (anterior
coelomic cavity) is linkedwith the outside via a canal. If, in fact,
hemichordates have obliterated a real original transformation
very completely from their life history, they may turn out not to
show any lateralised gene expressions orthologous to the
‘vertebrate-phylotypic’ ones of situs.
The settling of, for instance, a crinoid echinoderm larvamay
be recapitulating an ancestral 908 transformation when its
right-hand coelomic cavities degenerate on turning to face
the substratum, while the left ones collaborate with the left
ectoderm to surround the gut and generate the radially
symmetrical adult system.(32) Definitive information on the
gene expression signals that orientate formation of the adult
oral–aboral axis within the bilateral echinoderm larva will be of
great interest in relation to the vertebrate situs cascade. The
direction of torsion at larval settling, while interesting, is less
germane to the issue, because among extant echinoderm
groups, either the oral or aboral pole can be applied to the
substratum dependent upon the adult form and lifestyle.(33)
Amphioxus may represent a form which, while having
reacquired a largely fusiform adult body, still recapitulates
dramatically some steps in its morphological derivation from
thenon-symmetrical ancestor. Theascidian ‘tadpole’maybea
dispersal form, but is not larval in the sameprimordial sense as
the ciliated echinodermpluteus. It thus shows the anatomically
gratuitous left expressions of nodal and pitx as remnants of its
axially transformed origin. Vertebrates, according to this view,
havevery largely redevelopedananatomical symmetry in their
locomotory/ neural structure in adaptation to lifestyle. But, in
their visceral structure, as a heritage from the deep past, they
have retained the left–right (one-time dorsoventral) devel-
opmental organisation that is now positively co-opted for
the adaptive arrangements of situs. Fig. 1 outlines, in a way
divorced from any particular interpretation of fossil forms,
Hypothesis
416 BioEssays 26.4
such derivation of contemporary left–right structure via an
axial transformation from an ‘ur-bilaterian’ ancestor.
What are we to make of the contemporary vertebrate
mechanism of developmental symmetry breaking within an
ostensibly symmetrical embryo? It could be a de novo
invention, utilising molecular chirality reliably to directionalise
activation of the one-time dorsoventral, now to be left–right
dimension of organisation. This might be necessary because
the massive outer-resymmetrisation of the vertebrate body
has reached backwards into development, dominating gas-
trular structure and threatening to remove original cues for the
left–right axis. Alternatively, it may be more directly derived
from a directional settling mechanism whereby the still ur-
bilateral, ciliated larva of ancestral deuterostomes underwent
its 908 axial re-organisation to give the benthic adult. Intere-
stingly, the locomotion of some contemporary deuterostome
larval forms is spiral because of ‘off-axis’ ciliary beat, rather
than solely due to their hydrodynamic shape.(34)
Additionally, there is now striking evidence from a uro-
chordate(35) and a vertebrate(36) for a left–right differential
distribution of gene products, immediately postfertilisation,
that could be functionally relevant to polarisation of the phylo-
typic left–right gene expressions in the otherwise symmetrical
embryo. Such direct asymmetrical placement of proteinsmust
reflect a utilisation of molecular chirality in the mechanical re-
organisations that follow fertilisation in these forms. It is of
great interest that urochordates and the vertebrates each
exhibit modes of development that might be considered to
have by-passed entirely the ‘ur-bilateral’ truly larval form
primordial to the group.
The transient but clear, directional non-symmetry in bird
gastrular anatomymay be yet a further clue to the evolutionary
Figure 1. Schematic of a 908 transition within vertebrate ancestry. Three body forms are shown, each in dorsal plan and in composite
transverse sectional views (representing axial levels opposite dashed lines). The original bilaterian ancestor (left) has paired special sense
organs (black), filter- or detritus-feeding apparatus anteriorly, paired and probably segmented mesoderm structures (stippled), and a
tubulargutwithout left–right but possiblywithdorsoventral complexity.Shortlyafter theproposedmorphological transition (centre), thecase
is shown in which an original right side becomes ‘ventral’, and substratum-applied, with concomitant invasion of ventral surface
specialisations and loss of some subset of the original right members of paired structures. The result is a non-symmetrical body plan.
On the right is shown an arbitrary stage in the secondary re-acquisition of bilaterality in the locomotory and neural (sensory) structures, by
organ-pairing in the head, but perhaps by progressive re-rotation of the locomotory tail structure and its forward invasion along the dorsal
body wall, nowwith a notochord (see Ref. 24). The result is essential symmetry of the outer body wall, together with a developmental basis
for left–right structure centred in the viscera. This scenario takes account of evidence from comparative developmental anatomy of extant
forms, comparative gene expression and molecular taxonomy, but does not follow any detailed phylogeny proposed from fossil evidence.
Hypothesis
BioEssays 26.4 417
depthofvertebrate left–right structure.Noticedat least75years
ago and mentioned at intervals since, this has recently
received renewed attention.(37,38) As shown schematically in
Fig. 2, during several hours of development, the region fated
to give rise to the axial structure of the anterior body does not
give the appearance of a bilateral animal’s embryo. At these
stages, the notochord—traditionally mid-dorsal mesodermal—
is being derived entirely from deep tissue at the right of the
node, while the floorplate—traditionally mid-dorsal in the over-
lying neural plate—originates entirely from the more super-
ficial layer at the node’s left. This structure becomes apparent
well before ‘phylotypic’ lateralised expressions of nodal, Pitx
and Snail-related genes are in place, but late enough to be
developmentally downstream of early left–right ion transport
processes as discussed by Levin.(1) It is reversed in the right-
hand member when parallel twin axes form within one blasto-
derm, in keeping with the classical observation that situs itself
is often disturbed or reversed in thismember of experimentally
produced twin patterns.(39) In single embryos, the node
asymmetry is directionally robust, however, even though the
later asymmetries and gene expressions of situs can be con-
fused or reversed by subsequent experimental treatments.(37)
Due to lack of observations during the restricted relevant
stages, we cannot currently say how many other ‘blastoderm-
type’ vertebrate embryos, e.g. reptiles or even some mam-
mals, show such transient handed asymmetry.
The possibility of an ur-bilaterian dorsal–ventral to left–
right axial transformation deep within vertebrate ancestry has
not found wide favour, mainly because the fossil evidence
on which it was originally proposed seems impossibly fragile
and subjective to cellular and molecular developmental-
ists.(24,27,28,40) The transformation itself has been presented
as if it had occurred within one ontogeny to create a ‘hopeful
monster’ ancestor. While such a scenario is not unthinkable in
a remote earlymetazoanworld, themajority of current thinkers
about even macro-evolutionary change are of gradualist
persuasions. But any such transformation relevant to chordate
origins must have occurred back in the explosive Cambrian
phase of metazoan diversification, where fossil traces will
remain problematic. Its palaeontological originators should
be given full credit for the idea of a 908 transformation, but
evolutionary developmentalists at large should not be dis-
tracted from assessing the other independent evidence for
its occurrence, outlined above. The chordate, or extremely
chordate-like, vetulicolians (lower Cambrian, China) are a
plausible source of vertebrate ancestry,(41) but it is currently
unclear whether they display situs-like directional asymmetry.
908 axial transformations could even have occurred sepa-
rately in the ancestry of echinoderms (including ‘calcichor-
dates’) and of chordates, rather than in a common ancestor.
Summary and conclusions:
a differential evolutionary constraint?
Situs, the directional left–right structure within the vertebrate
body, is most obvious in the packing of the viscera but is in fact
more pervasive, (e.g. embryo torsion, and also form of the
human cerebral hemispheres(42)). It develops through initia-
tion of a left–right difference, across the midline of the early
Figure 2. The asymmetrical anatomy of the
avian (chick) gastrula. At left, the whole embryo-
forming area (area pellucida) of the blastoderm is
shown in dorsal view, anterior at top. Dashed box
indicates the central region, embracing the centre
of gastrulation activity (Hensen’s node) that is cur-
rently giving rise to anterior parts of the postgastr-
ular midline (head process). At right, detail of this
boxed region is shown, together with its transverse
section (below). The axial level of section is
indicated by the horizontal dashed line, and the
observer of the section faces anteriorly. Arrows
indicate indentations in the neurectodermal sur-
face 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 the gutter-like groove that passes
anteriorly to swing into the midline as the future
floorplate of the nervous system (shown cross-
hatched). The right-hand indentation flanks the
deep-lying mass of the nascent notochord, which
is positioned eccentrically to the right. The emer-
ging notochord (shown stippled) swings progres-
sively into place in the midline beneath the
floorplate as the node regresses from each level.
Hypothesis
418 BioEssays 26.4
embryo, in a signal that triggers mutually exclusive, propagat-
ing cascades of ‘left’ and ‘right’ gene expressions within
relevant tissue layers as formation of the body plan proceeds.
Left and right tissue characters are subsequently expressed
throughdistinctive contributions to growth andmorphogenesis
in asymmetrical organ systems. Understanding is currently
most advanced for the middle, ‘propagative’ sector, which is
essentially vertebrate-conserved, although the list of gene
interactions involved in the cascades continues to grow and
some of these may act independently in parallel. Our relative
ignorance concerning the ‘downstream execution’ sector
only tracks with current ignorance regarding ‘downstream
executive’ form-shaping mechanisms in organogenesis
generally (though see the Theoretical Morphology article in
this issue on pages 405–412(43)).
Understanding of the ‘initial symmetry-breaking’ sector is
currently at a particularly interesting andcontroversial stage.(1)
Evidence has been accumulating in several vertebrates for
the basic role, at stages preceding gastrulation, of vectorial
transport processes within the junction-linked intracellular
space, leading to ion-channel-driven electrochemical gradi-
ents across the embryo midline. In at least one vertebrate and
one protochordate (related outgroup) form, molecular compo-
nents of this intracellular symmetry ‘breaking’ system achieve
left–right differential distribution during the very first cleavage
divisions when the fate map for the axial body plan is being
orientated within the egg material, suggesting at least that
chordate left–right structure is evolutionarily deep. Together
with a wealth of less direct evidence from comparative devel-
opmental anatomy, comparative expression of gene ortholo-
gues and molecular phylogeny, this indicates that biologists
should take seriously the central (and non-original, for
example, Ref. 28) proposal of this article. This is that while
adaptive, in enabling optimal design for new lifestyle, verte-
brate directional left–right structure was not a latter-day
innovation. Rather, it was a preadaptation stemming from a
remote ancestral dorsal–ventral to right–left axial transforma-
tion, around which a newer, secondary bilaterality of the outer
locomotory/neural body has been built.
In addition to the structure of situs, complementarities of
function between anatomically equivalent regions in right and
left halves of the brain may also be uniquely advantageous,
and distinctive to vertebrates. These could enable an increase
in the diversity of parallel subprocesses that feed into the final
behavioural outcome of advanced cognition. The devel-
opmental information enabling such neural lateralisations
appears in some cases to be independent of that underlying
situs, but could also stem ultimately from vertebrates’ unique
deep history. Developmental relationships between neural
functional ‘handednesses’ and situs are reviewed in depth
elsewhere.(13,44,45)
An implication of this ‘axial transformation’ proposal is that
left–right encoding, in vertebrate development, may be of a
kind not found elsewhere in the animal kingdom. Thus neither
vertebrate-type left–right neural-functional complementarity
nor profound left–right differential anatomy may be attainable
among otherwise similarly complex protostome animals.
Being untransformedly bilaterian, these animals lack the
developmental basis for genetic tissue labelling of ‘leftness’
and ‘rightness’ as such, and with it the possibility for highly
controlled left–right differential expression of the geno-
me. A non-trivial example of differential evolutionary con-
straint,(see e.g. 46) deriving from historical contingency, may
thus have been in operation since the Cambrian metazoan
diversification. If conjectures regarding the adaptiveness of
left–right structure for vertebrate rates of energy expenditure
and lateralisations of higher cognitive function have any sub-
stance, that constraint has been quite consequential.
Certainly, in development of gastropod molluscs or nema-
todes, right–left differential organisation is propagated struc-
turally via a cleavage pattern of defined ‘handedness’. It is
probable that chiral information from a protein assembly
ultimately underlies such cases, and acts via relative cell posi-
tioning, as left–right adult structure can be reversed experi-
mentally by micromanipulation of the latter in the embryo.(47)
But no such forms have confronted, as successfully as have
vertebrates, the problemsposedbyhighly energetic lifestyle at
large body mass, or by complex cognition.
Genetic selection experiments on the ectodermal pattern-
ing of Drosophila have consistently failed to demonstrate that
any left–right encoding information exists to be harnessed for
controlled asymmetry.(e.g. 48) A very slight but real directional
asymmetry of wing size/shape, conserved among several
dipteran fly species, hasneverthelessbeen recently observed,
and claimedasevidence for an arthropod ‘left–right axis’ in the
sense that term has acquired for vertebrates.(49) But such
asymmetry must surely be functionally gratuitous, rather than
in any sense adaptive, and it seemsmore plausible to propose
some intrinsic directional biass in the accuracy with which
mirror-image shape can be generated, for certain kinds of
multicellular structure. The morphogenetic program of wing-
disc development through metamorphosis is very demanding
mechanically, involving collaboration among thousands of
ultimately flattened and extended cells, whose cytoskele-
tons are after all built of ‘handed’ or chiral self-assembling
molecules. Such a phenomenon, a small, biophysically driven
biass to shape-control originating in protein chirality, is quite
distinct froma left–right ‘axis’ of regionalised geneactivation in
the vertebrate sense. The contrast is illustrated abstractly in
Fig. 3. Striking but circumscribed directional asymmetries
occur sporadically among protostome forms with indefinite
cleavage patterns,(reviewed in 50) e.g. the directionality of the
cheliceral size asymmetry in certain free-living decapod
crustaceans. These may represent amplifications of such
minute biophysically driven shape inequalities, through their
subsequent genetic linkage to growth patterns. But without a
Hypothesis
BioEssays 26.4 419
general developmental ‘left and right’ encoding through gene
expression, such mechanisms appear to have been of limited
evolutionary potential.
Further test of this idea of differential phylogenetic con-
straint would come from systematic search for directional
neural-functional left–right complementarity in the brains of
cephalopod molluscs and eusocial insects, neither of which
utilise determinate cleavage plans in development. A pan-
adaptationist view might suggest that, if this can and has
been achieved in vertebrates, it should be present among
such animals, as indeed should a situs-like visceral packing in
fast-swimming squids. I currently know of no examples.
Figure 3. Two contrasting modes of developing directional left–right asymmetry in bilaterian animal forms. Each column of diagrams
represents, from top to bottom, aspects of the progression of development in an axial animal seen in plan view. Left column: thearrangement seen in vertebrates. At early stages, probably while the midline of patterning is itself being established, vectorial transport
processes establish a right–left differential situation across this midline with respect to some signal that initiates distinctive cascades of
gene activity (horizontal arrow concentrating an initially evenly distributed molecule). These cascades (solid black arrows) ‘label’
widespread components of the tissue structure with ‘left’ and ‘right’ identity in normal development (represented as filled or hollow nuclei),
and are stabilised by negative regulatory inter-relationships between their component genes and by a barrier to signal invasion within the
now anatomically discrete midline. This directly enables rudiments of paired organ structures to be assigned different numbers of founder
cells, and/or to undergo differential schedules of subsequent growth and morphogenesis, in effect, potentially separately evolvable left nd
right structures.Rightcolumn:a possible,more evolutionarily restrictedmechanism in protostomeanimals directly inheriting the original
bilaterian dorsoventral axis, and lacking a stereotyped cleavage plan that directly propagates molecule-based chirality. There can be no
differential left and right labelling of early tissue, so both founder-cell numbers and early growth schedules for paired organ primordia can
show only fluctuating, i.e. non-directional asymmetry dictated by the intrinsic limits of developmental control (represented here by those of
the author’s freehanddrawing control). Butwithin cells of an epithelial sheet, whichmight undergoa demandingprogramof vectorial growth
and force production in morphogenesis of complex shapes, an apicobasal structural polarity becomes defined. An intrinsic, molecularly
defined chirality of the cytoskeletal assembly will then bear an opposite relation to the forming supracellular structure on either side of the
midline. This could be sufficient to provide an unavoidable directional asymmetry to the results of mirror-image shape production (shown
exaggerated in the diagram, in relation to that empirically observed in, for example, the insect wing, Ref. 49). Downstream gene control
linkages, harnessed to this local differential result, could conceivably be utilised to achieve further differential development within the
structures concerned.
Hypothesis
420 BioEssays 26.4
Acknowledgments
I am grateful to Michael Akam, Max Telford, Simon Conway-
Morris, AdamWilkins and anonymous referees for comments
during the preparation of this article, though the views ex-
pressed are my own.
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