Mecanismos do desenvolvimento e evolução

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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 : jonathan.cooke5@btinternet.com)
(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 the
vertebrate 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. An
overview, rather than an exhaustive scholarly review, is given of recent advances in molecular understanding of
the mechanism that ensures normal development of ‘correct ’ situs. While the asymmetry itself and its left/right
direction are clearly vertebrate-conserved characters, data available from various embryo types are compared
in 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 gene
expressions, clearly exists. It now seems probable that earlier steps, in which symmetry-breaking information is
reliably transduced to trigger these cascades on the correct sides, are also conserved at depth although it remains
unclear exactly how these steps operate. Earlier data indicated that the initiation of symmetry-breaking had been
transformed, 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 visceral
packing 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 in
analysis 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 an
evolutionary novelty imposed on a true bilateral symmetry, at or close to the origin of the vertebrate clade. A
major purpose of this review is to advocate an alternative view, on the grounds of comparative anatomy and
molecular 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 adult
form in a vertebrate ancestor. Viewed this way, vertebrate or chordate origins are best understood as the novel
imposition 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 that
controls situs development. But the function of this particular diencephalic asymmetry is currently unclear. Other
left-right partitionings of brain function, including the much more recently evolved, cerebral cortically located
one associated with human language and hand-use, may be controlled entirely separately from situs even though
their directionality has a particular relation to it in a majority of individuals.
Finally, possible relationships are discussed between the vertebrate directional asymmetries and those that
occur 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 left
and 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 377
DOI: 10.1017/S1464793103006298 Printed in the United Kingdom
CONTENTS
I. Introduction: Scope and level of this review ........................................................................................... 378
II. 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 .......................................................................................................................................... 388
III. 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 or
independent 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 ......................................................... 399
VI. Conclusions .................................................................................................................................................. 400
VII. Acknowledgements ...................................................................................................................................... 402
VIII. References .................................................................................................................................................... 402
I. INTRODUCTION: SCOPE AND LEVEL OF
THIS REVIEW
Despite the nearly perfect bilateral symmetry of vertebrate
axial anatomy, paired sense organs and limbs, it is widely
recognised even by non-biologists that the major visceral
organs are packed into the human body cavity in a highly
asymmetrical 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 vast
majority of individuals. The rare human situs inversus
however, the complete mirror-reversal of normal visceral
anatomy, is a perfectly viable state of affairs (Torgerson,
1950), though the decidedly less rare heterotaxias, confusions
of left-right anatomy within individuals, are usually patho-
genic. It becomes clear, from comparative study of a range
of vertebrate embryos, that while the developed viscera and
particularly the heart and greatblood vessels have become
more complex in function and anatomy, the directionality of
the situs asymmetry is vertebrate-conserved.
Such directional asymmetry is quite distinct from the
phenomena of fluctuating asymmetry and antisymmetry. In
the former, limitations of ‘buffering’ of the developmental
mechanism against (genetically or environmentally con-
ditioned) perturbation limit the precision with which right
and left ‘ symmetrical ’ structures can be mirror replicas
within individual bodies. In antisymmetry, competitive
suppressive mechanisms ensure that right and left structures
are very different in size etc. (as in certain arthropod
appendage pairs), but with essentially random ‘handedness ’.
Certain more subtle directional asymmetries within ver-
tebrate structure are reliably co-ordinated with, and could
thus be considered part of, situs. The preferred direction of
axial torsion within the uterus or egg-shell, seen in most
types 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 boundaries
that is clearest in acraniates (Wada, Garcia-Fernandez &
Holland, 1999) but detectable in vertebrates ( J. Cooke,
unpublished observatons on chick and frog embryos), and
left-right differences in both developmental and adult
branchial anatomy of some agnathan vertebrates.
Less universal but still highly significant is the preponder-
ance (around 9:1) of right-hand-use preference for human
skilled activites ; the most conventional use of the term
‘handedness ’. This is strongly (but not universally) linked
with the phenomenon that most of us have the capacity for
language use preferentially located in our left cerebral
cortices, with other, harder-to-define but complementary
cognitive 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 higher
cognitive capacities within the hemispheres, although in fact
various ‘discordancies ’ among these are common and not
strongly dysfunctional.
Left-right functional complementarities of brain usage,
with a preponderant directionality corresponding to the
human one, have recently been found to characterise most
of the hominoid (human-ape) clade, and are probably
related to the onset of language-like cognitive abilities and
associated differential hand use (Annett & Annett, 1991;
Cantalupo & Hopkins, 2001). But in addition to this, there is
a whole range of phenomena involving differential function-
ing, sometimes linked to detectably different anatomy, of
the two sides of the brain in fish, reptiles, birds and non-
primate mammals (see Section IV and references). For most
of these, however, it is unclear whether population levels of
directionality are similar to those of human hand preference
etc., 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 itself
although this is not positively known.
Molecular understanding of developmental mechanisms
has recently made explosive advances for situs itself, and
these mechanisms, including data from all the experimental
‘model ’ vertebrate embryo types, have been comprehen-
sively reviewed for developmental specialists by several
authors (cited in Section II.1). The aim of the present article
is not to replicate or even update these reviews but instead to
survey the field for a broader readership, and in relation to
three key questions of evolutionary interest ; the degree of
conservation or universality of the whole situs developmental
cascade, the evolutionary origin of situs, and finally the
degrees of developmental and thus evolutionary relationship
between 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 set
of gene orthologues. These initiate the differentials in
growth and morphogenetic movement that characterise
visceral organ primordia on either side of the midline to
produce situs anatomy. But dramatic apparent differences in
the events that lead up to this stage, among vertebrate
embryo types, have led to suggestions that the earlier sector
of mechanism, that which breaks the embryo’s apparent
bilateral symmetry in the first place, has varied opportun-
istically as much as has pre-gastrulation anatomy during
vertebrate evolution. While acknowledging that this may
turn out to be the case, I nevertheless propose that current
data are consistent with in-depth conservation of even this
early, so-called symmetry-breaking sector of mechanism.
Recently, experimental gene expression data in zebrafish
(Danio rerio) have linked the origin of an anatomical brain
asymmetry, the diencephalon-associated habenular and
parapineal one that is probably of functional importance in
fish, amphibians, reptiles and birds, with the phylotypic situs
gene 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 necessarily
all linked with this particular feature ; they remain to be
fitted into what traditional developmental geneticists would
have referred to as a ‘pedigree of causes ’ underlying
vertebrate asymmetries. Current data seem to link together
human hand-use preference and higher cognitive left-right
partitioning, as being controlled probabilistically by the
same system of left-right developmental information (see
Section IV and references). But strikingly, the data also
indicate that this particular neural lateralisation system is
quite independent from that controlling situs (Torgerson,
1950; Kennedy et al., 1999; Tanaka et al., 1999).
Evolutionarily speaking, ‘explanation’ for a consistently
lateralised arrangement of vertebrate visceral packing is not
hard to find. Given the material possibility, evolution can be
expected to have simulated optimal engineering design, and
the argument that such an arrangement is indeed optimal
for the vertebrate will be laid out in Section III. But, with
the 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 thus
been 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 distinctive
lifestyle requirements of an organism at or relatively close to
the origins of the vertebrate clade.
The present article re-emphasises data from zoology,
comparative anatomy and comparative gene expression
studies that advocate a different evolutionary scenario.
These data suggest that a vertebrate ancestor possessed an
evolutionarily ancient, non-symmetrical organisation, but
secondarily re-symmetrised its locomotory, outer body wall
and, to a large extent, nervous system as an adaptation to a
new lifestyle. The developmental substrate of the previous
non-symmetry was however retained, for further elabor-
ation of its increasingly complex viscera. Viewed in this way
the vertebrate body plan is a secondary re-imposition of
bilaterality, in the form of an almost symmetrical locomotor-
skeletal outer body wall and paired brain structure, upon a
radically non-bilateral adult body plan in the precursor
animal (see references cited in Section III.2). According to
this scenario, that non-bilaterality is evolutionarily deep,
characterising a clade embracing most or all deuterostome
animals. It was itselfa secondary departure from original
bilaterality, the mode of axial organisation ancestral to all
metazoans other than sponges, cnidarians and possibly
ctenophores (the clade Bilateria : Finnerty & Martindale,
1998; Holland, 1999). Initial symmetry ‘breaking ’, in
vertebrate left-right development, is then seen as the reliable
re-evocation of the ancestral adult non-symmetry, within the
symmetrical cellular anatomy of the contemporary blastula/
gastrula. On this view, in most vertebrate embryos, the
relatively recently imposed locomotory/neural bilaterality
has come to dominate the structure of the pre-gastrula so
completely that the lack of mirror-symmetry inherent in
biomolecular structure – usually referred to as molecular
‘chiral ’ information – must be recruited to initiate the
ancestral gene cascades as what is now a left-right ‘axis ’.
Tantalising indications do exist, however, of a left-right
component at the outset of vertebrate development (see
Section III.2 and references).
II. OVERVIEW OF THE DEVELOPMENTAL
MECHANISM FOR VISCERAL SITUS
Fully detailed reviews of this expanding field are widely
available (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 involves
some detailed consideration of anatomy and molecular
developmental genetics ; readers interested in the wider
issues might follow the remaining material without this
section, especially by referring to Fig. 1.
(1 ) A vertebrate-conserved ‘phylotypic’
post-gastrular sector
It has become clear that activation of the transforming
growth factor b (TGFb)-related intercellular signal gene
Evolutionary origin of vertebrate left/right asymmetries 379
‘Activin’
signal
FGF8
BMP4
Shh
Vg
signal
nodal
signal
lefty
signal
flow at
node
Pitx2
(prolonged, extensive
‘left encoding’)
BMP4
signal
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 elongated
site of gastrulation as seen from above, with the axis being generated in an anterior (top of page) to posterior sequence from a
regressing 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 than
that shown. The earlier, near-midline ones in the chick occur around the node, before the onset of regression or during its earliest
stages. Heavy hatched arrows connecting bolder gene names represent positive control (activation) input ; light stippled connections
leading 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 an
essential conserved mechanism imbuing this region with left
identity in all studied vertebrate embryos (Levin et al., 1995;
Collignon, Varlet & Robertson, 1996; Lohr et al., 1998).
Gastrulation proceeds as a sequence, in which mesoderm
destined for successively more caudal regions of the body
plan moves into place as a middle cell layer ahead of the
closing blastopore (in amphibians) or regressing Hensen’s
node (avian and other ‘blastoderm type’ and mammalian
embryos). Left lateral nodal expression initially follows this
wave-like sequence, beginning with material lying within or
just posterior to the heart-forming region. The mRNA
expression, presumably followed by peak levels of nodal
signal, is dynamic and is down-regulated after relatively
brief periods at successive antero-posterior levels, to disap-
pear at a level near the regressing node after around 10
somites have segmented, for instance, in the chick. This
nodal signalling in turn activates transcription of Pitx2,
encoding a homeobox-containing transcription factor. Once
activated, left Pitx2 expression is both more durable than
that of nodal and spreads anteriorly and posteriorly beyond
the nodal domain that triggered it. Pitx2, whose left-right role
appears to be that of an executive control gene for Left
developmental 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 sharp
boundary maintained from the inflow (future atrial) region
up into the outflow or ventricular region until advanced
stages of looped heart morphogenesis. Over an extended
period as the body plan is laid down, Pitx2 expression
becomes activated at the origin of the left contribution to
successive paired or asymmetrically developing visceral
rudiments, such as those of lungs, stomach and other
intestinal 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 a
domain that also appears to extend and then move back in
a wave-like manner, but beginning slightly earlier than that
for left-lateral nodal. Thus having begun in the anteriorly
positioned, compressed cardiac territory, right lateral SnR
expression is last detected in the posterior heart inflow
region only, at around the 12 somite stage in chick. The
orthologous snail-related gene is known from the frog (Xenopus
laevis) and zebrafish (Danio rerio), though expression studies
that would reveal a distinctive right-lateral expression
component have not been published. It thus seems reason-
able to suggest that right-lateral expression of this gene
be considered as an additional conserved component of
the gene cascade for vertebrate situs. It should be noted
that nodal, Pitx2 and this snail-related gene each have
other, conserved but bilateral expression domains in early
vertebrate embryos. These expressions, some of which
have attested developmental roles, are under separate
control (see Isaac et al., 1997; Patel, Isaac & Cooke, 1999,
for SnR).
It appears that where transient RNA expression is
appropriate, as for the wave of nodal signalling, this is
achieved through an early self-activating component in the
transcriptional control, followed by a damping negative
control input from a co-activated or downstream-activated
gene product. Thus a sequence motif in the control region of
nodal suggests positive feedback (i.e. self-activation) from
signalling through the nodal receptor pathway itself, while
negative feedback occurs through the antagonistic activity of
the closely related ligand (or anti-ligand) lefty-2/antivin on
nodal signalling at the protein level (Adachi et al., 1999;
Norris & Robertson, 1999; Saijoh et al., 2000). The lefty gene
is expressed in a left-lateral domain that closely tracks and
slightly follows the nodal one, and is probably activated
directly by the nodal signal that it then goes on to quench.
Prolonged and stable expression such as is required for
the Pitx2 role, by contrast, is organised through initial
upregulation by the upstream nodal signal, followed by self-
maintaining and collateral positive maintenance inputs that
include a binding site for the Nkx 2.5 transcription factor
(Shiratori et al., 2001).
Initial activation of lateral mesodermal SnR (chick) is
bilateral, and could be considered a default state. Then with
variable rapidity among individuals, it is downregulated on
the left as nodal expression first appears there (Isaac et al.,
1997). Experimental right-sided nodalprotein expression
ablates normal SnR expression there, while antisense
treatment targetting SnR expression ultimately leads to
abnormal Pitx2 expression on the right (Patel et al., 1999),
suggesting that SnR normally helps to suppress Pitx2
expression there. Thus, while it is clear that left nodal
signalling positively activates Pitx2 transcription directly
(Shiratori et al., 2001), a further, indirect double-negative
control relationship may help to ensure spatial exclusivity
between the key snail-related and Pitx transcription factor
gene expressions normal to right and left sides.
Pitx2 null mutant mice exhibit right cardiac isomerism,
i.e. ‘ right-hand’ developmental character in heart precursor
tissue from both sides of the midline, although the situation
for more posterior viscera is less clear and may simply
correspond to a lack of lateralising function, causing
heterotaxia (Kitamura et al., 1999; Lin et al., 1999).
Targetted ablation of msna in mice is not informative since
the gene has other, prior essential developmental roles, but
chick 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, are
probably phylotypic, while the evidence that the right-lateral SnR (snail-related zinc-finger transcription factor) role is phylotypic
remains incomplete.
Evolutionary origin of vertebrate left/right asymmetries 381
the same experiments, antisense-mediated SnR disruption
that is too brief to cause detectable right Pitx2 expression
nevertheless randomises the direction of heart-tube looping.
This looping direction, normally to the embryo’s right, is the
first vertebrate-conserved, gross-anatomical manifestation
of situs to develop. Loop reversal in these latter embryos
is independent of the development of ‘ left ’ and ‘right ’
structural character in the tissue contributions to the tube
inner wall, which can remain normal (Patel et al., 1999).
Overall results of perturbing SnR function thus suggest a
dual role in left-right development. A direct role ensures
right cardiac looping and perhaps correct embryo torsion,
but this is separable from the gene’s ‘cascade’ role in
propagating right-hand character within tissue by ensuring
repression of Pitx2. Pitx2, by contrast, appears to have a
prolonged and widespread role, determining left-specificity
in tissues of successively developing organs.
A variety of data demonstrates that in addition to
negative regulatory linkages between ‘opposing’ genes,
barrier functions within the axial midline structures consti-
tute a major mechanism maintaining proper laterality of
expression domains normal to left and right during post-
gastrular development (Meno et al., 1996; Bisgrove, Essner
& Yost, 1999, 2000; Meno et al., 1999). The cellular
structure of the midline appears on anatomical grounds
alone to constitute a relative barrier to diffusing intercellular
protein signals ; the notochord, a rod of tightly packed
vacuolated cells with a tough matrix sheath, is tightly
associated in vivo with the overlying floorplate, a midline
strip of cells integrated into the neural plate but displaying
at early stages several molecular affinities with notochord.
There are also midline-associated gene expressions that
appear to have a ‘barrier ’ role, notably including a member
of the nodal-related lefty group (see above), whose proteins
may act as an antagonist ‘ sink’ to nodal signalling by a
mechanism equivalent to ‘dominant-negative’ interference
with nodal ligand. A lefty is expressed in a strip at the left-
hand edge of, or bilaterally in, midline structures of all
vertebrates examined. Disruption or non-formation of an
anatomically normal midline, whether as part of a mutant
phenotype or due to microsurgical or other early embryo
manipulations, predisposes embryos to heterotaxias with
bilateral 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-right
structure and downstream gene expressions in relation to
the probable timing and axial position, within post-gastrular
development, of midline barrier interruption (e.g. Bisgrove
et al., 2000). Additionally, proper negative feedback onto the
early, autocatalytic activation of lateral nodal expression
appears necessary for its normal left restriction. Thus in a
mouse lefty null mutant, heterotaxias are associated with
abnormally extensive and bilaterally spreading nodal RNA
expression (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 target
genes whereby Pitx2 (left) and the snail-like orthologues
(right), actually execute the modulations of morphogenesis
and growth rate that constitute ‘ left ’ and ‘right ’ characters
in relevant tissues. As regards heart loop morphogenesis,
there is evidence that differential microfilament function
and character of the extracellular matrix are involved
(Itasaki et al., 1991; Tsuda et al., 1996). One complexity is
that structures that come to be situated at right and left in
the heart rudiment after its looping, were originally specified
on the basis of relative antero-posterior ordering within the
straight heart-tube. Thus gene expressions characterising
particular parts of the formed heart, that might be
considered 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 retinoid
signalling has been identified in the stable maintenance of
the conserved post-gastrular left-right gene expressions, and
thus the development of situs (Chazaud, Chambon & Dolle,
1999; Zile et al., 2000), though this retinoid role does not
appear to extend back into the earlier, symmetry-breaking
phase that will now be discussed (Chen et al., 1996).
(2) Initial left-right symmetry breaking in earlier
development: apparent diversification in timing
and in upstream cascade steps
A sector of vertebrate development beginning late in
neurulation, and extending through the organisation of
pharyngeal arches and a heart tube, can be regarded as a
relatively conserved ‘phylotypic ’ anatomical stage, the
pharyngula (although see Richardson et al., 1997). By this
stage, the vertebrate-conserved left-right gene expressions
described above have become established in lateral meso-
derm, and they too can justifiably be referred to as
‘phylotypic ’. But vertebrate cleavage, blastula formation
and early gastrulation are much more variable, and until
recently, it has seemed that mechanisms of breaking
symmetry to initiate the phylotypic left-right cascades on
the correct sides may be as diverse, among vertebrate
classes, as is this earlier developmental anatomy. Compara-
tive information for this early sector is with few exceptions
confined to the widely used ‘model ’ embryo types : the
zebrafish (Danio rerio), the clawed frog (Xenopus laevis), the
chick (Gallus gallus) and the mouse (Mus musculus), plus
relevant human clinical genetic observations. Since this is
essentially one embryo per taxonomic class, with no
agnathans, elasmobranchs or reptiles, we cannot even be
sure that the differing timings of symmetry-breaking and
first lateralised gene expressions observed typify the respect-
ive classes ; such arrangements might be labile on an even
finer evolutionary scale.
The various versions of early development seen in
mammals are clearly amongthe most derived to be found
in vertebrates. Observations on early events in the mouse
will nevertheless be described first, since they seem to
illustrate most clearly the theoretical predictions that, if the
embryo 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’ in
having no plane of bilateral symmetry) molecular infor-
mation (Afzelius, 1976, 1985; Brown & Wolpert, 1990;
Brown, McCarthy & Wolpert, 1991; Almirantis, 1995). The
differing arrangements in the avian (chick) embryo will then
382 Jonathan Cooke
be described. Chick not only enjoys the longest list to date of
genes with left-right lateralised expressions en cascade, but
was the first organism in which the recent explosive growth
of molecular understanding began (Levin et al., 1995, 1997;
Isaac et al., 1997; Pagan-Westphal & Tabin, 1998). This
chick gene cascade extends back into earliest gastrulation or
before, and is accompanied by a consistent asymmetrical
anatomy at mid-gastrulation (Wetzel, 1929; Lepori, 1969;
Hara, 1978; Cooke, 1995). Incomplete pictures of early
events in fish and frog leave it currently unclear whether
they align more closely with chick or with mouse in their
earliest lateralised gene expressions. This state of affairs has
been proposed to indicate a genuine evolutionary oppor-
tunism or lability among vertebrate embryo types, in the
initiating mechanisms that precede the conserved ‘phylo-
typic ’ sector (Yost, 1999, 2001; Wagner & Yost, 2000). Such
evolutionary lability of ‘upstream’ cascade steps in relation
to a conserved, downstream sector in developmental
mechanism is seen elsewhere, for instance in sex determi-
nation, and possible reasons for its origin have been
discussed by Wilkins (1995, 2002).
Brown & Wolpert (1990) most clearly characterised the
requirements for a molecular source of left-right symmetry-
breaking information in an otherwise symmetrical embryo.
This would have the formal properties of, for instance, the
upper-case letter F. If molecules or molecular assemblies of
this type were to become aligned in cells, with respect to two
other dimensions of organisation or ‘axes ’ that the cells
shared, a third dimension of alignment would necessarily
be created. If the initial shared alignments were antero-
posterior and apico-basal, as could be the case in epithelial
structure near the midline in a gastrulating vertebrate
embryo, then the molecular basis could be initiated for an
organisational difference between cells to either side of the
midline, or for a left-right polarised transport via gap
junctions and the joint intracellular space. If an axis of beat
of embryo cilia were to become fixed at an angle to an
embryo-wide axial polarity shared by the epithelial cells,
through a tethering according to the ‘F-molecule ’ principle
of the known chiral molecular structure of ciliary basal body
cross-section, then net bulk transport towards the right or
the left of an anteroposterior midline axis in the epithelium
could in principle result. Alternatively, a chiral (spiral)
ciliary action with a particular (clockwise or anticlockwise)
‘handedness ’ could be determinedmolecularly.With certain
spatial arrangements in groups of such cilia near a midline,
net bulk transport to the right or left could conceivably
result, although in this latter case it is important to recognise
that the F-molecule principle is not involved, since individ-
ual ciliary assemblies are only aligned with respect to the
one, apico-basal cell polarity. The general principles of
embryo symmetry-breaking by ‘conversion’ from chiral
molecular structure, together with one idea for their possible
realisation via ciliary activity, are illustrated in Fig. 2.
It was proposed many years ago that normal ciliary
activity in the embryo might somehow be translated into the
correct directionality of visceral situs, based on the inherited
human 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 body
structure, including spermatozoa tails, are affected in this
particular syndrome, causing infertility and chronic airway
congestion and infection as additional aspects. But structur-
ally atypical cilia, the protocila, have long been known to
exist in early vertebrate embryos, distributed one per cell on
the apical surfaces of epithelia facing certain cavities. Their
motility and functional significance had been questionable,
but following a recent exciting series of reports, it is clear
that such cilia are indeed motile. Gene products that are
necessary for their structure and normal motility are also
demonstrably required for normal symmetry-breaking in
situs development, at least in mammals.
A densely distributed group of protocilia on the ventral
surface of the mouse embryo node (anterior tip of the
primitive streak), creates in vivo a net flow toward the left
across the embryo midline (Nonaka et al., 1998). Normally
this must occur within the narrow extracellular space that is
the homologue of the primitive vertebrate archenteron. This
occurs during the middle part of gastrulation including the
start of node regression or head process formation. In
mouse, the midline notochord population derived by
ingression at the node is at first included in the epithelial
surface of the future foregut roof, and for some time
protocilia remaining on these cells continue densely to
populate the emerging midline anterior to the regressing
node. These cilia may also function to maintain the leftward
flow or, alternatively, may contribute to left-right barrier
formation (see Section II.1). Ciliary activity somehow
creates net flow to one side of the midline.
A variety of targetted null mutant mice, lacking function
of particular proteins involved in ciliary construction and
activity, show either absence or paralysis of the embryonic
protocilia in conjunction with gross disturbances of down-
stream ‘phylotypic ’ left-right gene expression and visceral
situs. One of these corresponds with the previously known iv
(inversus viscerum) mutant, the gene product now being
identified and characterised as left-right dynein (LRD), a
relative of the ‘axonemal ’ dynein subfamily (Supp et al.,
1997, 1999). In both mutant versions, where stiff, paralysed
protocilia are reported though other cilia are normal, the
predominant phenotype is random assignment of embryos
to 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 transcription
factor HNF4 (Chen et al., 1998), have no recognisable
protociliary structures. In these, interestingly, heterotaxias
may predominate, with absent or abnormal bilateral
expressions of ‘phylotypic ’ left-right genes, rather than
normal or reversed ones.
These correlations between ciliary activity and its known
molecular basis, the demonstrated extracellular ‘nodal
flow’, and symmetry-breaking for visceral situs in a mam-
mal, have undoubtedly impressed the community as a major
insight into mechanism. They indicate that reliable net
Evolutionary origin of vertebrate left/right asymmetries 383
Fig. 2. Possible conversion modes from molecular chirality to left-right developmental information. Upper two diagrams illustrate
how a macromolecule or molecular assembly showing chirality (properties formallysimilar 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 the
differential cascades of the lateralised gene expressions.
Once started correctly, these are maintained as exclusive by
their regulatory cross-relationships and by the midline
barrier function (see Section II.1). Since the nodal gene is
already locally active around the base of the regressing node,
and indeed in the mouse this transcription normally
becomes progressively enhanced on the left edge (Collignon
et al., 1996), a direct working hypothesis would have nodal
protein itself as the leftwards-translocated ‘morphogen’ (see
Hamada et al., 2002). This idea has the added attraction that
regulatory relationships between nodal and its immediately
downstream and related gene lefty appear to embody the
principles of local self-activation (by nodal protein) leading
to longer-range lateral inhibition (by lefty protein). These
have long been proposed on theoretical grounds to charac-
terise symmetry-breaking morphogen systems, whose be-
haviour when computer-modelled can amplify a small initial
disturbance or inhomogeneity into a major, stable asym-
metry in the spatial distribution of a developmental signal
concentration (Turing, 1952; Gierer & Meinhardt, 1972;
Gierer, 1981).
Nonaka et al. (2002) now claim to have demonstrated the
direct action, in mouse at least, of fluid flow across the
relevant embryo surface in determining the directionality of
the ‘phylotypic ’ gene expressions and of situs itself. The
strikingly ingenious experiments involved culture of the
embryos in a microchamber, in which medium flows of
controlled rates could be imposed from either left to right or
right to left in relation to the primary embryonic axis. The
flow rates that reversed situs of normal embryos, or could
determine that of ivx/x embryos (with inactive cilia), were
grossly comparable with those caused by the normal ciliary
activity. Interference was most effective in the period
immediately preceding normal onset of left-lateral nodal
gene expression. At the same time, the authors emphasise
the incompleteness of our understanding of the physical
basis of the endogenous directional flow, which appears
literally to cross the field of beating cilia rather than, for
instance, being whirlpool-like with a particular ‘handed-
ness ’ around the node. If the beat of individual protocilia is
simply spiral, the net leftward flow would need to result
somehow from their dense distribution coupled with the
antero-posteriorly distinctive shape of the node base. In this
way chiral molecular structure with only one axis of
alignment, 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 directionality
because its beat has an off-axial in addition to a spiral
component, as speculated above, the ciliary basal body
structure could indeed be embodying the full ‘F ’ principle of
Brown & 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 of
cells that are developing epithelial structure, within which a
bar-like thickening due to the piling up or ‘columnarisation ’
of cells comes to define an axis, the primitive streak. As in
the mouse, the definitive postgastrular midline arises as cells
ingress through the shortening streak to form the new
endoderm and mesoderm layers, the space beneath defini-
tive endoderm ahead of the node being equivalent to
primitive archenteron. These arrangements have obvious
structural homology with those in the mouse, but the chick
node is a less focussed structure and built on a more massive
cellular scale. The mouse ‘ turret ’ structure with clear upper
neurectodermal and basal endodermal epithelial layers (see
Bellomo et al., 1996) is not found. As gastrulation enters the
node-regression stage in chick, the notochord occupies a
new middle layer from its inception, without transient
integration into the midline archenteron (foregut roof).
Single protocilia exist on many cells over an extensive,
diffuse region near the midline, throughout gastrulation, on
the apical (i.e. outer) surfaces of both endodermal and
upper, neurectodermal layers. They are sparsely distrib-
uted in space on endoderm due to the flattened, stretched
and heterogeneous nature of these cells, and this situation
persists once node regression has started (Manner, 2001;
Essner et al., 2002; J. Cooke, unpublished observations).
This renders implausible the production of significant
directional flow as seen in the mouse, especially if ciliary
beat has a spiral component. The apical neurectodermal
protocilia in this central region are somewhat more
prominent, but it should be recognised that any structural
‘handedness ’ within the anchored molecular assembly of
cilium and basal body itself, when viewed from a particular
surface of the embryo, would necessarily be reversed as
between the apical cell surfaces of endoderm and neurecto-
derm. To suppose anything else would be to deprive this
molecular assembly of the crucial ‘conversion’ role (Brown
& Wolpert, 1990) that is the whole attraction of a proposed
association of ciliary action with the origin of tissue level
left-right asymmetry.
transport (arrow from left to right) to initiate differences across the axial midline. Lower digrams illustrate two different ways in
which chiral structure inherent in cilia at the basal surface of an embryo midline region, shown facing the reader, could create net
flow 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 two
dimensions within cell structure according to the ‘F-molecule ’ principle. Net directional flow results in some way from co-operativity
between such cilia in relation to the antero-posterior architecture of the embryo’s node region. The right diagram shows the
situation where the chiral structure within individual cilia allows for anchoring at a particular angle to the embryo’s long axis, so that
their intrinsically directional, whiplash-like beat produces the net leftward flow. It remains unclear, at the time of writing, which
principle is most used in the real situation.
Evolutionary origin of vertebrate left/right asymmetries 385
In addition to the above problems, a peri-nodal flow
comparably timed with that seen in the mouse would be
redundant as an initiator of lateralised gene expression in
the chick. A cascade of such expressions, preceding any
known for mouse, bridges between the earliest gastrula or
even 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 are
indeed diffusely present on the chick nascent archenteron
surface, around the time that lateralised gene expressions
are first detected in early gastrulation, these associated
lateralised gene expressions are quite different from thosein
mouse (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, then
opposite the anterior streak to the right. This occurs before
the 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 prior
step in the cascade is probably the right-sided accumulation
of such a ligand, perhaps by preferential gene expression.
Selectivity relationships between known receptors and
ligands within this superfamily are incompletely known, so
despite a report of preferential right activin bB mRNA
expression at the same early stages (Levin et al., 1997), it
remains unclear that the ligand involved has been identified.
Experimentally, activin protein perturbs downstream left-
right gene expression and situs when applied to chick
blastoderms during these early stages (Levin et al., 1995;
Isaac et al., 1997), but experiments may not have been
sufficiently controlled to ascertain that perturbation distinc-
tively follows from a left-hand experimental application.
Recombinant activin protein is highly diffusible, and as will
be seen, further roles for other ligands of the TGFb
superfamily, in addition to the well-understood one of nodal
itself, 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 emerges
ahead of the regressing node. Early on, in chick, this
expression usually appears more prominent in the presump-
tive neural ventral midline (floorplate) than in underlying
chordamesoderm. Soon, it becomes significantly extended
back into the node wall on the left side only. Experiments
indicate that it is the preferential right-activation of the
‘activin pathway’, just described, that normally represses
Shh expression to the right of the node. Chick nodal
expression arises shortly afterwards in deeper-lying, meso-
dermal structure immediately lateral to the Shh expression in
the left node, and this clearly corresponds with the left-
accentuation within the bilateral ring of nodal expression at
the base of the mouse regressing node. Though this near –
midline left nodal expression is maintained for some hours in
both types, no cascade role for it is evident experimentally in
chick. Instead, the left-lateral Shh signal at the node directly
evokes the separate, broad left-side ‘phylotypic ’ nodal
expression via a double-negative mechanism, involving
activation 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 to
counteract, on the left, the otherwise widespread bilateral
bone morphogenetic protein (BMP)-4 signalling at this
stage. This in turn relieves a pre-existing BMP-4 repression
on nodal expression, thus involving yet another member of
the 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 Shh
expression at the right in the node, activates fibroblast growth
factor (FGF )-8 expression in the right anterior flank of the
streak behind the node (Boettger et al., 1999; Schneider et al.,
1999). FGF-8 signalling in turn can repress Shh, and
activates the transcription factor gene NKX3.2 in the right
streak flank as well as the right-lateral ‘phylotypic ’ SnR (see
Section II.1). The potential complexity is indicated by the
capacity of BMP-4 signal, whose gene is intensely activated
at right of the normal node, to repress normal Shh and
activate ectopic FGF-8 expression when locally applied at
left (Monsoro-Burq & Le Douarin, 2001). Additionally, at
around the time of these differential Shh, FGF-8 and BMP-4
expressions, the specific adhesion-mediating molecule N-
cadherin is expressed in an asymmetrical pattern around the
node and anterior streak (Garcia-Castro, Vielmetter &
Bronner-Fraser, 2000). Role relationships are not yet clear,
but interestingly, effects of blocking N-cadherin function on
heart-loop direction and embryo torsion may be mediated
without affecting the otherwise key downstream ‘phylo-
typic ’ Pitx2 and SnR expressions. This raises the possibility of
parallel independent, and not interlocked, gene expression
cascades.
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 application
before stages of node regression, doubly right-sided, doubly
left-sided, left-right reversed and normal versions of the
downstream gene expressions are produced in approxi-
mately equal numbers of embryos (Isaac et al., 1997). This
suggests failure of symmetry-breaking followed by a recov-
ery process, in which repressive regulatory links ensure that
either ‘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 the
inactivations of LRD or ciliary kinesins in mouse (Collignon
et al., 1996; Nonaka et al., 1998; Marszalek et al., 1999).
It has been hard to establish whether embryo types
represented by zebrafish and Xenopus laevis fall more into line
with mouse, in proceeding directly from a symmetry-
breaking mechanism to the phylotypic cascade, or with
chick in utilising an intermediate cascade of lateralised but
‘near-midline ’ gene expressions. Attempts to investigate
this in zebrafish through microinjection of Shh-expressing
or -interfering constructs are hard to interpret, since the
gene has two potential roles in left-right development. In all
vertebrate embryos it supports normal function of the
notochord and floorplate and thus of the midline signalling
386 Jonathan Cooke
barrier, in addition to any left-specific signalling role it may
have. Thus in mouse, where no functional or expression
evidence for a left-specific Shh role exists, the Shh null mutant
phenotype is now recognised to show left-right anomalies
connected with breakthrough expression across the midline
of the ‘phylotypic ’ nodal and Pitx2 expressions (Meyers &
Martin, 1999; Tsukui et al., 1999). To date, no evidence
suggests that the cascade just described in chick, beginning
with right-hand accentuation of an ‘activin pathway’ and
proceeding through left midline Shh expression via a caronte-
like function to the left phylotypic cascade, is utilised by frog
or fish.
We have seen that the derivation of left-right organisation
from chiral structure (Fig. 2), i.e. symmetry-breaking by
molecular ‘conversion’ (Brown & Wolpert, 1990) occurs
considerably 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, by
contrast, such cilia occur in Xenopus laevis (frog) and Danio
rerio (teleost fish) embryos only at late gastrular or even
‘pharyngula ’ stages, and at posterior axial positions (Essner
et al., 2002). Especially in the fish case, the timing and
position seems hard to link with an origination of the
lateralised gene cascades through archenteron-located flow.
(3 ) Is there a unitary mechanism for
developmental origin of vertebrate situs?
Further problems of interpretation remain, for the simplest
view of a vertebrate-universal, initial symmetry-breaking
mechanism via leftward ciliary flow. The iv gene product
LRD, despite its familyrelationships, is largely distributed
cyoplasmically and in regions outside those where active
protocilia occur, including an anatomically left-lateralised
component, in the gastrula/neurula (Supp et al., 1999). Such
a distribution would not be consistent with a role confined to
initial symmetry-breaking by molecular conversion. The
possibility exists that LRD, and/or some of the other gene
products whose requirement for normal symmetry-breaking
has been assumed to follow from their functions in cilium
formation/activity, have additional functions in polarised
transport machinery within the joint (gap-junction linked)
intracellular space. These transport functions, directionally
tethered within cells on the ‘F ’ principle (Brown & Wolpert,
1990), could also be relevant to symmetry-breaking by
molecular ‘conversion’. An explanation may still be
required for the apparently different consequences of LRD
loss on the one hand (random situs correlated with paralysed
cilia) and loss of other proteins such as kinesins on the other
(heterotaxia and absent cilia), although a larger database
would 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 at
population level rather than randomising it or causing
heterotaxias. Elucidation of the nature and cell function of a
protein whose mutational alteration achieves such a reversal
has been eagerly awaited, during the gene’s protracted
positional cloning since its initial characterisation. ‘ Inversin ’
turns out to be a large, intracellular protein of unclear
relatedness, containing a domain of multiple ankyrin-type
structural units believed to signify tethering to other proteins
(Mochizuki et al., 1998; Morgan et al., 1998). The mutant
phenotype also exhibits early lethality due to kidney
malfunction, possibly signifying inversin involvement in
transcellular transport mechanisms (see reference below to
Ca2+ signalling), but characterisation of the cellular func-
tion remains elusive.
Crucially and paradoxically, inv null mutant mouse
embryos do not exhibit reversed nodal ciliary flow. Instead,
a possible subtle alteration in shape of the node base, and a
somewhat more turbulent flow of reduced efficiency in
leftward 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 their
model to incorporate and explain this finding, but at the cost
of depriving the model of its original merit, which was as an
explanation for de novo symmetry-breaking. The model
might nevertheless be rescuable from these paradoxical
observations if, for instance, it were ultimately to turn out
that some intimate cellular architecture of the basal node or
nearby midline was instrumental in the normal leftward net
flow. The inv mutant alteration, a partial deletion within the
protein structure, might conceivably alter this in a way that
failed to reverse the experimentally observed flow but did
reverse an in vivo functional equivalent. It must be pointed
out, however, that the published report of manipulations of
the flow does not include results of the formally appropriate
‘negative control ’ condition, namely a manipulated absence
of net flow (Nonaka et al., 2002). Such controls are
particularly desirable in view of the known tendency of
culture in flowing-medium conditions to randomise situs
development in mouse and indeed in other vertebrate
embryos (Brown et al., 1991; Fujinaga et al., 1992; Fujinaga,
Lowe & Kuehn, 2000; J. Cooke, unpublished observations
on chick and blastocoel-irrigated Xenopus laevis embryos).
Meanwhile, evidence has accumulated for the role of
directional intracellular transport via gap junctions, in
symmetry-breaking at the earliest stages of both chick and
Xenopus laevis development (Levin & Mercola, 1998a, b,
1999; Levin et al., 2002). Experimental interference with
gene expression for a connexin that is a principle compo-
nent of embryo gap junctions, and related pharmacological
channel interference, results in downstream randomisation
of situs and associated gene expressions in both species. An
intercommunication barrier is reported to exist, for both
frog blastula and chick blastoderm, at positions that could be
taken as ventral within the respective fate maps. Under these
circumstances, if transport machinery had been aligned on
‘F-molecule ’ principles in relation to the embryo axis,
preferential ‘morphogen’ transport could occur across a
presumptive dorsal midline to build up differences between
right and left. The linking of frog and chick embryo types by
evidence of this sort is significant because they appear
otherwise divergent, within the range of more downstream
cascade steps, since the latter but not the former exhibits the
near-midline sequence of local gene expressions that starts
early in gastrulation and culminates in left Shh and Caronte
(see Section II.2). Observations on mouse, where the claim
Evolutionary origin of vertebrate left/right asymmetries 387
for causal primacy of ciliary flow seems strongest, further
support a generality of the involvement of intracellular small
molecule transport in the symmetry-breaking phase for situs
(Pennekamp et al., 2002).
A final recently forged link between apparently diverged
embryo types concerns the distinctive left-right role of the veg
(vg) genes, encoding yet another subgroup of the TGFb
superfamily of signal proteins. Vg1, the first-identified
member in Xenopus laevis, has been a strong candidate for
an endogenous maternally encoded signal whereby the
yolky vegetal blastomeres induce mesoderm and definitive
endoderm in the equatorial zone of the blastula (Thomsen &
Melton, 1993; Kessler & Melton, 1995). Orthologues and
closely 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 for
primary axial patterning roles has perhaps been usurped by
the nodal genes themselves (Varlet et al., 1997; Varlet,
Collignon & Robertson, 1997; Brennan et al., 2001), even in
the yolky eggs of frog and fish where maternally translated
genes are prominent at early stages (Schier & Shen, 2000). A
developmental patterning role for Vg genes is nevertheless
suggested by their special processing requirements for
release of active ligand. The enzyme system, whereby
functioning C-terminal polypeptide is cleaved off from the
pre-proprotein at secretion, is not the widely available one
utilised by TGFb family members generally. Normal
development in the frog and the chick occur without
production of immunologically detectable amounts of the
working Vg ligand, despite abundant bilaterally distributed
mRNA and pre-proprotein. The assumption is that the
endogenous role(s) of Vg genes involve localised production
of the minute amounts of ligand required, due to localisation
of a crucial part of the processing machinery.
Experimental ectopic mis-expression of Vg genes employs
chimaeric DNA or RNA constructs, in which the C-
terminal distinctive Vg sequence is fused with an N-terminal
sector encoding the permissive processing parts from
another 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; Hanafusa
et al., 2000). Applied on the right, Vg is able systematically
to reverse the expression of the ‘phylotypic ’ cascade and
morphological situs itself, rather than randomising these or
giving rise to left-isomeric development as experimental
right 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 thandue to
direct right-ectopic activation of the response pathway to
nodal protein, for instance. This has led to a proposed
endogenous role as a ‘ left-right co-ordinator ’ signal in
normal development. The implication is that this role is
normally exerted from a relatively lateral left position. The
endogenous time of action is unknown, but data suggest that
experimental action on situs is still fully effective in the early
gastrula stage, and possibly much later than this (Toyoizumi,
Mogi & Takeuchi, 2000).
Either chick Vg, or chick or mouse nodal proteins have
been locally mis-expressed on the right, in an extensive
series of chick blastoderms in culture ( J. Cooke &
S. Withington, unpublished work). Here, too, Vg exerts a
distinctive effect. Whereas Nodal protein, present on the
right from stages of node regression and midline formation,
randomises situs and induces right ectopic expression of nodal
and Pitx2 genes as would be expected, right-expressed Vg
protein is able with significant frequency to reverse the
expression of Pitx2 to give right expression only. Further-
more, as would be expected from its downstream position in
the normal cascade, right-ectopic nodal protein is unable
ever to disturb the left-sided Shh expression at the node itself
(or the associated gastrular structural asymmetry, see
Section III.2). Comparable ectopic Vg protein expression
does indeed disturb or reverse these features, when imposed
from stages before they have developed. Finally, the null
mutant phenotypes have been described for the mouse Vg1
orthologue Gdf-1 (Rankin et al., 2000), and for furin, a
convertase component of a processing pathway that may
distinctively be required for certain TGFb superfamily
ligands (Constam & Robertson, 2000). These involve right
isomerism and left-right inversions of particular visceral
arrangements, and typically, absence of the ‘phylotypic ’
left-lateral gene expressions. These observations all suggest
that Vg genes have a vital vertebrate-conserved role, situated
not far downstream of the initial symmetry breaking process
for situs.
(4) Changing left-right co-options of gene
orthologues during vertebrate diversification,
and other puzzles
FGF-8 in chick has a distinctive right-hand expression which
is 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 comparable
stages (Meyers & Martin, 1999). The transcription factor
gene Nkx 3.2, a left-hand player apparently downstream of
nodal and kept repressed by right-hand FGF-8 in chick,
correspondingly appears to swap sides in mouse (Schneider
et al., 1999). In chick, as also described in Section II.2, the
extracellular 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 phylotypic
left-lateral nodal expression by interfering with BMP-4
signal. In mouse, where Shh has no known lateralised
expression or role and where a left caronte gene expression
orthologous with that of the chick has not been found, a
gene cognate with caronte, Dte, is nevertheless reported to
show distinctive right-hand expression at comparable stages
in the node (Pearce, Penny & Rossant, 1999). This is
puzzling for an additional reason; potentiation, rather than
counteraction, of BMP-4 signal is believed to be important
at a right-lateral position, as this antagonises Vg signal there
(see above, Ramsdell & Yost, 1999; Branford, Essner &
Yost, 2000). Most tested Cerberus-Dan family proteins
antagonise BMP-4 signalling although dte has not explicitly
been tested.
In any version of a lateralising gene cascade, there is no
reason why a particular gene should not have distinctive
roles in different steps on opposite sides of the body, pro-
vided that the cascade steps are sequential, circumscribed in
388 Jonathan Cooke
time and well-regulated within the developing anatomy.
BMP-4, for instance, might have more than one successive
role on the right, counteracting Vg signalling from the left
in all embryo types (Ramsdell & Yost, 1999; Branford
et al., 2000), and restricting Shh expression at the node to the
left in types utilising this expression (Monsoro-Burq & Le
Douarin, 2001). Additionally, it has a later one on the left
in connection with the heart in at least one embryo type
(Chen et al., 1997). It is partly for this reason that negative
interference or targetted mutagenesis of receptor and
downstream signal transduction components can be prob-
lematic in interpretation when there is a ‘ left-right ’ pheno-
type. Each deleted component may have had normal roles
through signalling by more than one ligand within a
superfamily, and could thus have acted in ‘right ’ or in ‘ left ’
propagation, or in symmetry breaking, at different times. A
recent report concerning FGF-8 in rabbit establishes that
apparent ‘ side-swapping’ of gene roles between embryo
types can occur even within a vertebrate class (Fischer,
Viebahn & Blum, 2002), and may be more dependent upon
the morphology of gastrulation than upon deeper evolution-
ary change. This paper also clearly indicates the interpret-
ative perils attending multiple successive roles for particular
genes within one overall ‘cascade’.
Despite striking apparent differences, observations cur-
rently leave room for the belief that both the left-right
developmental gene cascade and the underlying mechan-
isms of deriving symmetry-breaking information from chiral
molecular structure, might be conserved for vertebrates.
Protocilia probably are present, albeit in a wider and less
dense distribution, across the archenteron or equivalent
surfaces of all vertebrate embryo types over a prolonged
period of early development. Pending an exact understand-
ing of the way in which these cilia operate, the possibility
remains of their broadly conserved role involving directional
extracellular transport. But there is the strong additional
possibility that some early process of intracellular polarised
transport is universally present, at pre-gastrula or later
stages, and functionally important in symmetry-breaking for
all vertebrate types. There is a need to elucidate the role of
the inversin (inv) protein. Additionally, a process at or close
to the head of the cascade also appears universally to in-
volve the left-lateral Vg signalling, while other TGFb signal
superfamily members have widespread roles. It is note-
worthy that this superfamily of signal proteins, centrally
involved in vertebrate axial induction and anteroposterior
organisation (Smith et al., 1990; Cooke, 1991; Schier &
Shen, 2000; Brennan et al., 2001), should be central also in
the mechanism of situs.
III. VIEWPOINTS ON THE EVOLUTIONARY
ORIGINS OF SITUS
(1 ) The functional ‘ just-so’ story: situs as a
vertebrate (chordate) invention
It can plausibly be argued that, provided a basis for its
mechanism existed within cellular organisation, a reliably
directional packing of the major viscera within the stream-
lined body wall would have been strongly selected for during
the origin of modern vertebrates. The argument runs as
follows. Transition from a sedentary or crawling, detritus- or
filter-feeding, to an actively swimming predatory lifestyle
is likely to have accompanied the origin of the vertebrate
clade. Greatly increased metabolic demands would have
been involved. Additionally, a square–cube relationship
applies between available surface areas for nutritional
absorption and gas exchange on the one hand, and tissue
mass on the other, if strictly equivalent anatomical structure
increases in linear size. If, as seems likely, a progressive size
increase took place, there would therefore have been strong
selective pressure for proportional increase in the lengths
and complexity of tubular viscera and ofpumping vascu-
lature. Within the streamlined outer ‘ locomotory’ body
shape that was meanwhile being selected for, such increases
for rate of food absorption and of vascular exchange could
only have been accomodated by the more complex looping
and/or coiling of these organ systems. For gut function, the
relationship is intuitively obvious, while in the case of
vascular function the effect of a spiral and looped arrange-
ment, in optimising the pumping output per unit mass
of muscular tube wall, has recently been described for the
mammalian heart (Kilner et al., 2000). Provided that the
visceral packing problem is ‘solved’ in precisely the same
way in all normally developing individuals, the further
opportunity arises for intimate co-ordination, thus subtle
co-optimisation, between the originally quite independent
peristaltic actions of vascular and digestive systems.
Given a bilaterally symmetrical cellular structure of the
gastrula, and the disparate origins of the internal organ
rudiments within the body plan, regularisation of internal
packing could only occur if cascades of gene expressions,
propagating separately in tissues to either side of the
midline, imbued them with left and right positional
identities. In this way, left-right differential growth and
morphogenetic patterns could be initiated co-ordinately in
the 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 lateralised
gene expression cascades. As detailed in Section II.2,
Brown & Wolpert (1990) made an influential formulation of
the problem in asserting that the only mechanism whereby
such 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 that
indeed some such molecular conversion process is needed if
the embryo starts out bilaterally symmetrical at the cellular
level of structure. Thereafter, appropriate regulatory exclus-
ivities between the lateralised gene expressions that are
triggered, together with a stabilising barrier function in the
midline against cross-invasion of signals, will ensure geneti-
cally ‘ left ’ and ‘right ’ sides with potentially separately
evolvable developments.
Among vertebrate embryo types examined to date, the
majority indeed appear entirely bilaterally symmetrical at
stages before the first features of situs develop. The bilaterian
clade of animal forms, by definition, is considered to have
Evolutionary origin of vertebrate left/right asymmetries 389
Fig. 3. Schematic of a proposed dexiothetic transition within vertebrate ancestry. Three body forms are shown, each in dorsal plan
and in composite transverse sectional views (-axial levels indicated by dashed lines). The original, truly bilaterian ancestor on the left
has paired special sense organs (black) and filter- or detritus-feeding apparatus anteriorly, paired and probably segmented mesoderm
structures (stippled), and a tubular gut without left-right but possibly with dorso-ventral complexity. The central diagrams represent
the 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 of
true bilateral symmetry. Most recent accounts of the
explosion of molecular knowledge about vertebrate left-
right development have, understandably, been from mol-
ecular genetic researchers whose project is a mechanistic
understanding of contemporary embryonic development.
They have tended to lack exposure to the disciplines of
comparative anatomy and phylogenetics, with their accent
on the dimension of historical contingency in evolution and
their propensity to ask: ‘via which route do current
biological arrangements happen to have come about? ’ It
has therefore been easy to propose or assume that a
symmetry-breaking mechanism, and the gene-expression
cascades for situs, were ‘ invented’ entirely in response to the
selective pressures attending the ancestry of vertebrates
themselves, or at least that of the clade of most obviously
related organisms in which they are embedded, namely the
chordates. The exciting correlation of mutations that affect
ciliary structure or function with disturbance of situs and of
the ‘phylotypic ’ left-right gene cascades in mammals (see
Section II.2), has brought the feeling that an understanding
of this molecular ‘ invention’ is close at hand. The whole
mechanism seems to conform with what theory would
dictate as necessary, during each contemporary vertebrate
embryo’s development, for directional de novo breaking of a
‘primordial ’ bilateral symmetry.
(2 ) A constrasting view: situs as the co-optation
of 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 bilaterian
sort. Rather, it is seen as having evolved a secondary,
derived bilaterality in the outer ‘ locomotory’ body wall and
nervous 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 of
diversification of animal forms, abandoned the primordial
symmetry ancestral to the Bilateria in a profound morpho-
logical transformation. In essence, the ancestral bilaterian
right-hand side is proposed to have become a new ‘ventral ’
surface applied to the substratum, with resultant loss of
primary 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 termed
dexiothetism. It may have occurred with the foundation of
the entire clade known as the Deuterostomia, or alterna-
tively, be shared by a more restricted grouping that includes
echinoderms and chordates (see below). Vertebrates would
on this view retain a deeply embedded, non-symmetrical
‘visceral ’ body organisation resulting from the dexiothetic
transformation, combined with an axial but only secondarily
re-symmetrised ‘ locomotor ’ component. The archaic com-
ponent including its regionalised developmental gene ex-
pressions, laid out in a defined way with respect to this newly
re-symmetrised organisation, would provide the basis for
any retained, then further elaborated, internal left-right
structure. Dexiothetism is illustrated in outline in Fig. 3,
together with its proposed consequences for vertebrate
structure.
Hemichordates, echinoderms, urochordates, cephalo-
chordates and vertebrates rather clearly form a clade, the
now-accepted Deuterostomia, when fossil (stem) forms,
anatomical structures shared by extant forms, features of
embryonic development and the newer (mostly 18S ribo-
somal DNA-based) molecular systematic evidence are all
considered together (Gislen, 1930; Jefferies, 1975; Jefferies,
1986; Jefferies, 1991; Jefferies, Brown & Daley, 1996;
Cameron, Garey & Swalla, 2000; Holland, 2000). The
Hox-type homeobox gene cluster seems basal to animal
axial organisation, and the possession of a relatively
complete cluster of Hox gene orthologues even by echino-
derms, that have no apparent axial bilaterality in their
extant 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 power
however, such that almost every group named above can
appear to be a basal sister group to all the others if particular
genomic sequences are considered