Breaking the left–right axis:do nodal parcels pass asignal to the left?Dominic Norris*

SummaryInmammals, left–right symmetry is broken by amechani-cally driven leftward flow of liquid at the embryonic node(nodal flow). Various models have emerged explaininghow this may happen. Work from Tanaka and collea-gues(1) has provided a new mechanism by which nodalflow may be breaking symmetry. They describe smallmembrane-bound particles, which they term nodal vesi-cular parcels (NVPs), that are carried to the left side of thenode. In the paper, they argue how signals carried withinthese parcels may break L–R symmetry. BioEssays27:991–994, 2005. � 2005 Wiley Periodicals, Inc.

Introduction

While externally mirror symmetrical between left and right, all

vertebrates show an evolutionarily conserved left–right (L–R )

asymmetry in the placement and patterning of the internal

organs and vasculature. Yet early vertebrate embryos are

initially morphologically L–R symmetric, raising the question

of how L–R symmetry is broken in a consistent manner during

vertebrate embryogenesis. The first morphological signs of

the breaking of symmetry are seen in the looping of the

primitive heart tube (8.5 days after fertilisation in the mouse),

but this is preceded by molecular asymmetries. It has been

known for some time that symmetry in the mouse is initially

broken at a transient embryonic structure called the node. In

an exciting recent paper Tanaka and colleagues have pro-

posed a new mechanism by which symmetry may be being

broken.(1)

The emergence of models

In 1990, theoretical work fromBrown andWolpert(2) examined

how L–R symmetry might be broken in a consistent manner;

they discussed how a chiral molecule could theoretically

translate anteroposterior and dorsoventral information into

left-right information. Subsequent molecular studies over the

past 10 years have described asymmetrically expressed

genes and defined a genetic pathway connecting them

(reviewed in Ref. 3). In all vertebrates studied to date, the

signalling molecule Nodal is expressed in the left lateral plate

of the embryo activating a signalling cascade that results in

left-sided expression of the transcription factor Pitx2. This

nodal signalling cascade acts downstream of activity at a

structure called the node. While work in the chick revealed an

upstream cascade of asymmetric gene expression at the node

culminating in left-sided node expression of nodal, sonic

hedgehog (Shh) and its receptor patched (Fig. 1A), no similar

cascade was detected in the mouse. Indeed another mechan-

ism emerged. Analysis of Kif3b null embryos,(4) in which L–R

patterning is randomised, led to the discovery of motile

monocilia within the node. These cause a leftward flow of

liquid across the node and this ‘‘nodal flow’’ was postulated to

break L–R symmetry. Though initially controversial, many

mutants affecting cilia structure or function were found to

affect L–R patterning. A series of elegant embryo culture

experiments have now clearly demonstrated that an artificial

nodal flow can direct the nodal signalling cascade to either the

left or right side of the embryo.(5) Although it is now generally

accepted that nodal flow mechanically breaks L–R symmetry,

the question remains of how the flow acts. Initially it was

suggested that nodal flow carries a morphogen to the left

across the node, signalling specifically to the left side of the

node.(4) Such a morphogen would have to be short-lived to

prevent the signal from recycling to activate the entire node. A

variation on this model emerged when it was demonstrated

that Nodal in the node is required for left-sided Nodal

expression.(6) As Nodal protein is able to activate the nodal

signalling cascade and squint (a zebrafish nodal) had been

demonstrated to be able to act at a distance,(7) it seemed

possible that Nodal itself might be being carried to the left

lateral plate of the embryo. However, it has never been

established that nodal flowcould carryamolecule to the lateral

plate.

Another model of how nodal flow might break L–R

symmetry emerged from the study of Pkd2.(8) The resultant

‘‘two-cilia model’’(8,9) argues that two populations of nodal cilia

MRC Mammalian Genetics Unit, Harwell, Oxfordshire OX11 0RD, UK

*Correspondence to: Dominic Norris, MRC Mammalian Genetics Unit,

Harwell, Oxfordshire OX11 0RD, UK. E-mail: d.norris@har.mrc.ac.uk

DOI 10.1002/bies.20309

Published online in Wiley InterScience (www.interscience.wiley.com).

BioEssays 27:991–994, � 2005 Wiley Periodicals, Inc. BioEssays 27.10 991

Abreviations: Shh, sonic hedgehog; Ihh, Indian hedgehog; NVP, nodal

vesicular parcels; Smo, smoothened; L–R , left–right; RA, retinoic

acid.

What the papers say

exist; the motile cilia previously described and a second

population of non-motile Pkd2 containing mechanosensory

cilia. In this model, the motile cilia, predominantly in the pit of

the node, rotate to cause nodal flow which is detected by

mechanosensory cilia in the periphery of the node. The

direction of nodal flow results in left-sided mechanosensation,

which is translated into calcium signalling on the left of the

node. This model was in part based on work demonstrating

that renal cilia containing Pkd2 can release intracellular

calcium in response to mechanical stimuli, induced either by

direct mechanical stimulation or by fluid flow.(10)

Nodal vesicular parcels

In a recent paper in Nature,(1) Tanaka and colleagues describe

another mechanism by which nodal flow may break L–R

symmetry. They describe the release of small membrane-

bounded vesicles from the floor of the node, which they term

nodal vesicular parcels (NVPs). Using scanning electron

microscopy, they visualised NVPs at the cell surface of node

cells, apparently in various stages of release and with

transmission electron microscopy shortly before release from

the cell. Ultrastructurally NVPs appear to comprise multiple

lipophilic granules bounded by a membrane. Live imaging of

labelled nodes revealed the releaseof smallmembrane-bound

particles from the node at the rate of one every 5–15 seconds.

Thesewere carried across the nodeby nodal flow (from right to

left), hitting the left wall of the node where they fragmented.

This resulted in the obvious transfer of phospholipid (and

presumably NVP contents) from the floor to the left side of the

node. In immotile cilia mutants, as might be predicted for

embryos lacking nodal flow, NVPs were still released but did

not move quickly to the left, instead being carried slowly in

Figure 1. Breaking left–right symmetryat the node.A:A leftward fluid flow (indicatedbyarrows) is theearliest knownasymmetric event in

mammalian left–right determination, resulting in activation of the nodal signalling cascade in the left lateral plate. In contrast in the chick, a

cascade of asymmetric gene expression at the node is upstream of the nodal signalling cascade. B: FGF signalling in the node permits

nodal vesicular parcel (NVP) production. Nodal flow carries NVPs to the left side of the nodewhere they fragment, releasing lipid, SHH and

RA, activating left-sided Ca2þ signalling.

What the papers say

992 BioEssays 27.10

either direction by Brownian motion. Intriguingly, when they

examined Ca2þ signalling in immotile cilia mutants, they de-

tected bilateral Ca2þ signalling, in contradiction to the

predictions of the two-cilia model. In cilia-free mutants, NVPs

were again produced and, in the absence of nodal flow, did not

move specifically to the left; however, they appeared longer

lived than in immotile cilia mutants. This argues that cilia are

involved in the destruction (fragmentation) of NVPs on the left

wall of the node.

In the chick,Fgf 8 is expressed to the right but not left side of

the node and when misexpressed can repress the left-sided

nodal signalling cascade.(11) In contrast, study of a hypo-

morphic allele of Fgf8 in mouse implicates FGFs in promoting

left sidedness.(12) Tanaka and colleagues used both pharma-

cological and biochemical methods to block FGF signalling,

demonstrating that interfering with FGF signalling abolishes

the normal left-sided Ca2þ signal in the nodes of cultured

embryos. These treatments also blocked NVP production. Yet

by adding either Indian hedgehog (IHH), retinoic acid (RA) or

SHH (all factors present symmetrically within the embryonic

node), they were able to rescue calcium signalling at the node.

Intriguingly, SHH and RA specifically rescued left-sided Ca2þ

signalling, while IHH stimulated bilateral Ca2þ signalling.

When NVP production was examined, they found that SHH

and RA rescued production of NVPs that were carried

leftwards by nodal flow. Intriguingly IHH did not rescue NVP

production, suggesting that IHH specifically stimulates Ca2þ

signalling while SHH and RA activated left-sided Ca2þ

signalling through their rescue of NVP production. Using

antibodies to SHH and RA, they detected both SHH and RA in

NVPs being released from the floor of the node. They further

reported seeing a higher level of SHH protein on the left of the

node than the right in just under half of the embryos that they

examined.

Together these data suggest a model (Fig. 1B) whereby

FGF signalling in the node is required for the production of

NVPs. The precise role of SHH and RA in NVP production is

unclear, although it can rescueNVP production in the absence

of FGF signals. NVPs carrying RA, SHH and possibly other

molecules are carried to the left side of the node where they

fragment on contact with cilia. This thereby asymmetrically

distributes factors, which were made within the pit of the node

and loaded intoNVPs, to the left side of the node. The resulting

signalling at the left side of the node breaks L–R symmetry

within the embryo and (by mechanisms yet to be understood)

results in left-sided Ca2þ signalling. This is presumed to

activate the left-sided nodal signalling cascade.

A new paradigm

As with most very novel findings, this work both provides

possible new explanations and raises many questions. In

someways, themodel is an evolution of the original idea that a

morphogenmade within the pit of the node is carried to the left

side of the node. If we simply replace the morphogen with

NVPs that are destroyed at the left side of the node, we have a

one-way street for the signal and it cannot recycle across the

node. It further presents amechanismexplainingFGF function

in mouse L–R determination. Significantly, this work may also

consolidate aspects of our understanding of the stepupstream

of the left-sided nodal signalling cascade between mouse

and chick. Asymmetric Shh expression at the chick node is

both necessary and sufficient to induce the left-sided nodal

signalling cascade;(13) however, no asymmetric hedgehog

expression has ever been reported in mouse. NVPs as pre-

sented in this paper, provide a mechanism that could deliver

asymmetric hedgehog signalling at the mouse node. While

suchaconfluenceofmechanismsbetweenmouseandchick is

intellectually highly appealing, it must be remembered that

there is currently no evidence for asymmetric hedgehog

signalling at the mouse node. Indeed patched-1 expression

(usually upregulated by hedgehog signalling) is perfectly

symmetrical at the mouse node,(14) arguing against the

reception of asymmetric hedgehog signals. The ability of

IHH, but not SHH, to bilaterally activate Ca2þ signalling is also

difficult to explain. Bothmolecules are capable of activating the

L–R pathway in chick,(15) albeit IHH less efficiently than SHH.

Both are expressed at themouse node and loss of both genes,

or of their common receptor smoothened (Smo), results in loss

of the left-sided nodal cascade.(14) From this it would be

possible to imagine that there is a loss of Ca2þ signalling at

the nodes of Smo null embryos. Whether or not NVPs are

produced in such embryos also remains to be determined.

Loss of the Shh gene results in predominantly bilateral Nodal

expression due to effects on the midline(16) rather than at

the node, a result that is not compatible with SHH being the

sole L–R morphogen in NVPs. The precise relationship of

hedgehog signalling and NVPs will presumably emerge from

future work.

The detection of bilateral Ca2þ signalling in an immotile cilia

mutant is striking in that it casts doubt on the current

incarnation of the two-cilia model. Yet models often evolve

over time. Indeed the NVP model itself leaves unanswered

questions about the role of molecules that are understood

within the context of other L–R asymmetry models. While far

beyond the scope of an initial paper, it remains to be seen how

the NVP model can integrate the phenotype of pkd2 null

embryos and their lack of any detectable Ca2þ signal at the

node that is central to the two-cilia model. The role of Nodal

expression at the node will also need to be addressed.

However the NVP model could explain the function of asym-

metric Lplunc1 expression.(17) Lplunc1 encodes a putative

lipid binding/transfer protein that is more highly expressed on

the left than the right side of the node, the same side that

accumulation of labelled lipid was reported in this analysis. It is

conceivable that Lplunc1 may be involved in lipid recycling at

the node.

What the papers say

BioEssays 27.10 993

Prospects

A number of questions will now need to be asked, particularly

whether NVPs are present in other vertebrates. Nodal cilia

have been found in all vertebrates studied to date(18) and nodal

flow has now been demonstrated in the zebrafish,(19) rabbit

andmedaka fish(20) aswell as themouse. It remains to be seen

how broadly NVPs are conserved through evolution. In this

study, the authors use asymmetry of Ca2þ signalling to assay

the breaking of L–R symmetry. While mutant analysis has

provided absolute correlation of a left-sided nodal Ca2þ signal

with left-sided activation of the nodal signalling cascade, it

remains unknown how the left-sided nodal signalling cascade

is activated. In the absence of such understanding, it remains

to be demonstrated that manipulations such as those used

in this analysis do affect the nodal signalling cascade as

predicted.

Acknowledgments

I would like to thank Aimee Ryan for discussion of ideas and

JennyMurdoch andRachael Hardisty-Hughes for reading and

commenting on the manuscript.

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