Vertebrate left and right: finallya cascade, but first a flow?Jonathan Cooke

Summary

Vertebrate development gives rise to systematic, normally reliably coordinatedleft-right asymmetries of body structure. This ‘‘handed asymmetry’’ of anatomymust take its ultimate origin from some chiral molecular assembly (one exhibitingno planes of symmetry and thus, having an intrinsic ‘‘handedness’’) within the earlyembryo’s cells. But which molecules are involved, how is their chiral propertycoordinately aligned among many cells, and how does it ‘‘seed’’ the differentialcascades of gene expression that characterise right and left halves of theembryo? Recent molecular characterisations of mouse mutations that randomiseor reverse body asymmetries have offered tantalising clues to the chiral initiatormolecules, but the findings in a subsequent Cell paper (Nonaka S, Yosuke T,Okada Y, Takeda S, Harada K, Kanai Y, Kido M, Hirokawa N. Randomisation ofleft-right asymmetry due to loss of nodal cilia generating a leftward flow ofextraembryonic fluid in mice lacking KIF3B motor protein. Cell 1998;95:829–837.[Reference 1]) may help us understand how the first gene expression asymmetriesoccur. BioEssays 21:537–541, 1999. r 1999 John Wiley & Sons, Inc.

Development of the consistent departures from bilateralsymmetry in vertebrate anatomy is now known to involvespecial, phylogenetically conserved cascades of gene activityproper to each side of the gastrulating embryo (reviewedin(2,3)). These cascades occur once gastrulation is underway,while the three-layered structure and antero-posterior axis ofthe body are generated. The signals involved are held apartand stabilised against left-right cross-invasion by the emerg-ing axial midline structure, which may also itself contribute tosignal propagation.(4) But this leaves unsolved the problem ofhow left-right symmetry might initially be broken, always withthe same handedness relative to the recently establishedplane of the future midline. It seems impossible to avoid theconclusion that the ultimate source of handed asymmetry ingross anatomy must be a chiral intracellular molecule ormolecular assembly, even in embryos like those of highervertebrates where the position and orientation of a body planonly becomes established relatively late within a large cellpopulation. A further excellent and phylogenetically more

wide-ranging review compares the development of asymme-try in the few-celled snail and nematode development styleswith that in vertebrates.(5) In the latter, chiral molecularstructure would need to become aligned, probably coopera-tively within many cells, in relation to the future anteroposte-rior dimension and to one other—say, the ‘‘top-to-bottom,’’ orapical-to-basal one within an epithelial cell layer. Until twoprior dimensions have been thus defined, left and right canhave no meaning on a supramolecular scale.

Macromolecular assemblies based around the helicalscaffold of microtubules, with their motor and polarisedtransport functions, are surely those that first come to mind ascandidate ‘‘seeds for handed asymmetry.’’ Human syn-dromes that include disturbed body laterality are associatedwith widely disfunctional and structurally abnormal cilia,(6) anda mouse mutant in the transcription factor HNF (hepatocytenuclear factor) 4 has no cilia at all, and randomised organasymmetry as well.(7) Excitement and speculation have re-cently been fueled by molecular characterisation of twomouse genes whose mutation had been known to disruptmajor organ asymmetry(8–10); iv (for inversus viscerum) andinv (for inversion of embryo turning). Both mutations actrecessively, with iv homozygotes showing a largely indepen-dent randomisation of the normally correlated body asymme-tries, whereas inv mice tend strongly towards total reversal. It

National Institute for Medical Research, The Ridgeway, Mill Hill,London NW7 1AA, UK. E-mail: j-cooke@nimr.mrc.ac.uk

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is as if each organ system has an inbuilt dynamic to goasymmetric, but there has been a loss of the fundamentalhanded information that coordinates them in the former miceand a reversal of it in the latter. Individuals of both mutantsshow appropriate misexpression (bilateral, absent, or re-versed) of prominent mouse members of the lateralised genecascade, placing the gene action points upstream of thiscascade.(11–13) The gene products have thus been candidatecomponents of the hypothetical chiral molecular apparatus,or of a process that first derives lateralised gene expressionfrom it.

The iv gene indeed turns out to encode a dynein, amicrotubule-associated force-producing protein, allied in struc-ture to the axonemal dynein subfamily normally found inciliary or flagellar structure. The large protein encoded by invprobably functions intracellularly, and shares ankyrin repeatdomains in a form particularly like those of the nematodeUnc44 gene product and thought to mediate tethering be-tween cytoskeletal and cell membrane-associated mol-ecules. But we cannot claim as yet to have much idea of invfunction. To propose its involvement in appropriate orientationbetween components of cytoskeleton and force-producingapparatus seems a reasonable speculation. But any attempt,at this point, to rationalise how this protein’s massive trunca-tion or absence(9,10) might systematically reverse globalleft-right information goes inappropriately far beyond data.

Although iv mutant mice appear not to have general orwidespread ciliary disfunction, these molecular findings haverevitalised speculation regarding the distinctive cilia-like organ-elles, so-called primary or monocilia, detectable on theventral surface of the mouse organiser region (centre of thegastrulation process) and on cells of the newly emergeddefinitive embryo midline just ahead of this.(14,15) The iv gene

(whose encoded protein is known as left-right dynein) isexpressed particularly in just these regions. Our modellingpropensities are now further titillated by perhaps the mostintriguing observation to date,(1) following on the knockout ofyet a further protein involved in organisation of microtubule-based structures. Mice deprived of the kif3B product, akinesin involved in microtubule-dependent transport that isevidently required in cila/flagella construction itself, entirelylack these monocilia of the organiser and early midline. Eventhough development as a whole does not reach advancedstages, it is readily ascertainable that major body asymme-tries are randomised in these mice.

The conversion process in asymmetry;the role of the organiserBrown and Wolpert,(16) in their very clear analysis pointing outthe near-necessity of a molecular origin for handed asymme-try, point out how some mechanism must effect a ‘‘conver-sion,’’ from coordinately orientated molecular structure withinsimilar cells to asymmetrically regionalised gene expression.Conversion must be an intercellular vectorial process, whichwould define a future left and right side of the embryo on thelarger scale at which genes begin to be activated differentlyon the two sides. These and other authors(17,18) have givencandidate conversion mechanisms, such as the build up of aleft-right concentration gradient in a signal molecule by itspolarised transport across tissue, or a left-right ‘‘rectifying’’asymmetry of molecular transfer properties in gap junctions.These ideas are pictorially represented in the most abstractpossible way in Figure 1A. After all, the only other a prioriplausible origin for chirality would be the Coriolis force due tothe Earth’s rotation, whose use is probably forestalled by itsbeing too weak for reliable perception by mechanisms on the

oFigure 1. A: Orientation of a structure for ‘‘conversion’’ of handedness from the molecular to the tissue level. (Adapted from concepts in (16)). Amacromolecule or molecular assembly present in one or more copies in each of one row of cells of an early embryo, and having the properties ofthe letter ‘‘F,’’ i.e., no planes of symmetry, might initially be orientated randomly within and between the cells (left diagram). The ‘‘F’’ is given ‘‘frontand back’’ faces of different character to emphasise that such a biomolecule could have differential sites for tethering to other cell structures onall major faces. Following the achievement of an apico-basal epithelial structure by the cells, and the definition of an anteroposterior axis ofpattern in the neighbourhood of the ‘‘organiser’’ (right diagram), the molecules could become coordinately orientated and be involved in apolarised transport process bringing about large scale left-right differences across a midline. B: The Right-hand twin rule. After division hasproduced an epithelial cell sheet (blastoderm) during bird development, twin organisers and embryonic axes occasionally arise instead of thenormal single one. Where these are ‘‘opposed’’ in orientation and arise at distant parts of the blastoderm periphery (left diagram), each axisderives its own normal left-right asymmetry of structure. This means that no global alignment of subcellular structures, underlying conversion toa macroscopic left-right flow or polarity, predates the organisers and axes (as in arrowheads from left). Were this the case, one emerging axiswould then necessarily acquire the reverse of the normally handed asymmetry. By contrast, when organisers of twin axes come to lie relativelyclose together in anteroposterior ‘‘parallel’’ configuration, the right but not the left member is frequently confused or reversed in handedness ofstructure (and gene expression). See text for the implications of this ‘‘right-hand twin susceptibility’’ rule. C: Possibilities for conversion frommolecular chirality of cilia to anatomical handedness. A group of cilia emerging from the ventral (basal) surface of epithelial cells at a particularposition along a pre-established midline produce a vortical flow component, as in the mouse embryo nodal cilia observed in(1). Its ‘‘handedness’’could reflect the chirality of the basal body (and ciliary) structure, and be susceptible to modulation either naturally or in absence of particularmolecular components (see e.g., (8)). The leftward or rightward components of this flow across the midline could ‘‘convert’’ nearby symmetricallypositioned concentrations of extracellular signals into lateralised ones, thereby breaking global left-right symmetry. Note that for suchmicrotubule-based structures and activities, unlike the formal situation with ‘‘F’’ molecules (A), only one (the ‘6’) molecular axis needs aligning ortethering within the cell to allow a ‘‘conversion’’ process.(16)

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scale of embryos. And anyway, international cricket andtennis (let alone surgery) would then have incorporated theadditional challenge that biological biasses in handed asym-metry would be reversed across the equator!

Vertebrate embryos, as stated earlier, delay the decisionsthat restrict the fates of their cells for too long for defined celllineage or asymmetrical cell division, with the obvious oppor-tunities provided by the microtubule- and motor molecule-organised mitotic apparatus, to be utilised in founding asym-metrical anatomy. Nevertheless, the structural and probablelineal relationship between mitotic spindle centrioles and thebasal bodies of cilia/flagellae has provided a tempting line ofspeculation. Being constructed on a basis of helical microtu-bules, these structures’ ‘‘plus’’ and ‘‘minus’’ ends becomealigned in relation to a particular surface of the cells in anepithelium, constructing cilia/flagellae those beat may has achiral (rotary) component. So, having observed their protocilia-free embryos and knowing the role of kif3B homologues inother systems, Nonaka et al.(1) re-examined directly theactivity of normal mouse ‘‘nodal’’ monocilia. After using aningenious method for direct observation, they find these ciliato beat with a rotary or vortical motion, producing in theseauthors’ particular micro-assay a dominant local flow to thefuture embryo’s left across the ventral surface at the posteriornode.

We now have a fairly good idea of when and, critically,where in development the initiation of lateralised gene-expression occurs. Examination at early stages of naturally orexperimentally induced twinned development, mainly in birdand frog embryos,(19–21) has produced the following informa-tion, schematised in Figure 1B. In ‘‘opposed’’ twin patterns,leading to head-to-head axes that have originated from pointsas far away as possible in the cell sheet derived by celldivision from the original fertilisation, each axis derives anormally handed asymmetry for its own embryo. Thus,chirality of cell structure cannot become (or be maternallygiven as) universally aligned throughout the cleavage cellpopulation to give a right and left at the outset. In theabnormal circumstance of two such opposed embryos thenoriginating, one of these would necessarily derive the ‘‘wrong’’or reverse-of-normal handedness. It is therefore consideredthat formation of each axis, with its own antero-posteriordimension, activates its own conversion process, whichprobably therefore occurs in the neighbourhood of the classi-cal vertebrate ‘‘organiser’’—the amphibian dorsal blastoporallip, or anterior end of the primitive streak or Hensen’s node inbirds. But when two organiser regions come to lie side by sideeven if separated by considerable tissue distance, with theirantero-posterior dimensions in ‘‘parallel’’ configuration, thensome interactions take place. Remarkably, these tend toconfuse or reverse the outcome specifically in the right-handmember, with the left-hand one being more robustly normal.These rules can be revealed by in situ analysis for various

early ‘‘handed’’ gene expressions, but for clarity in the figure,are shown with reference to the handed anatomical structureof Hensen’s node in birds. This intimate but clear andnormally reliable asymmetry precedes the onset of the morewidely accepted heart looping by many hours, as the earliestanatomical one observable in a vertebrate. This strengthensthe impression that conversion gets underway in the vicinityof the organiser and affects events fairly rapidly. The earliestlateralised gene expressions so far detected are in fact at thenode-forming region, a few hours before this anatomicalasymmetry,(19,22) and at a time very much corresponding withthat of the newly observed ciliary current in mouse. This is anintriguing tie-up even if the exact gene expressions constitut-ing the first part of the left-right cascade turn out not beconserved as between chick and mouse.(11) So how likely is itthat this observation represents a direct viewing of Brown andWolpert’s(16) ‘‘conversion’’ process?

Potential pitfallsThe claim of this current or ‘‘flow,’’ to be at least a rather directreflection of the conversion process in the overall mechanismof vertebrate left-right asymmetry, is therefore strong. Butpitfalls exist at various levels, beginning with an infelicitouschoice of term by the authors. New readers in this fieldbeware; they term their flow ‘‘the nodal flow,’’ understandablyin view of its location but confusingly since the nodal gene’sexpression is indeed among the first to be specificallylateralised in mice and other vertebrates, and is indeed to thenode’s left beginning shortly after they observe their ‘‘flow.’’But the conversion step is most unlikely to be a cilia-drivenleftward flow of an extracellular activator of nodal. In birdembryos at least, some earlier lateralised gene expressionsare seen and have been demonstrated to be causally up-stream of nodal’s left-specific expression, and among theearliest are some to the right.(19,22,23) Whether or not we haveyet observed the real first asymmetrical gene signal, resultingfrom the conversion process, the earlier-mentioned ‘‘righthand twin’’ rule seems to predict that this should be on theright of the organiser. As evident from the right diagram ofFigure 1B, only in this circumstance would just the right-handmember of a pair of parallel axes experience an abnormallybilateral initial signal, and thus perhaps initiate two ‘‘right’’-side gene cascades. The right-hand member’s real left sidewould be unprotected from the left hand member’s right sideby any midline barrier! So how about a progressive leftwardconcentration of an extracellular inhibitor near the node, toleave the first ‘‘asymmetric’’ gene itself activated on the rightonly? Once again, the right hand of a pair of nearby ‘‘parallel’’nodes should then confuse events around the left one, ratherthan vice-versa as observed.

In fact, these difficulties of detail are unimportant at thisstage, as are the interesting comparative problems that thenode in bird and reptile embryos is very differently shaped

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from that in mouse (see details in Nonaka et al.(1)), and facesonto a narrow intercellular cavity rather than the roof of thefuture gut. Figure 1C shows how a cluster of vortically beatingcilia in the midline could, in principal, convert initially symmetri-cal but anteroposteriorly patterned sources of diffusible sig-nals into left-right asymmetrical distributions. If such a signalor signals activated or repressed genes, the required ‘‘conver-sion’’ process from molecular chirality would then haveoccurred through alignment of microtubule-based structureswith respect to two prior global axes; the epithelial apico-basal one universal in the earliest embryo, and a patternedmidline structure defining the future body’s dorsal midline. Butis this indeed the required breakthrough? The long-recognisedcorrelation, of left-right instability with several independentlycaused abnormalities of ciliary function, certainly makes thenew observation exciting and suggestive. To be a plausiblecandidate signal to be moved into a concentration landscapearound the midline by such ciliary action, an extracellularmolecule would need very low effective diffusibility, perhapsbeing entrapped near the cell surfaces within the ‘‘greater cellmembrane’’ or glycocalyx. But this is thought necessarygenerally, for growth-factor like small proteins that pattern theembryo. The quickest way to add force to Nonaka et al.’sproposal, on which they must surely be already engagedcollaboratively, is to observe their cilia and flow (if any) in ivand, especially, inv homozygous mice. A reversed vortex andflow in the latter would be nice indeed. But until the putativeflowing morphogen were identified, there would still be roomfor the alternative notion. All these lesions may be disruptingthe formation or performance of cilia in correlation withdisruption of some other, orientated intracellular assemblyprocess in which the molecules are also involved. Thatstructure could itself underlie a conversion mechanism oper-ating, for instance, by left-right biased gap junction transport.

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