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First published online 3 May 2006
doi: 10.1242/dev.02384
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Review |
Developmental Genetics Group, Graduate School for Frontier Biosciences, Osaka University, and CREST, Japan Science and Technology Corporation (JST), 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan.
* Author for correspondence (e-mail: hamada{at}fbs.osaka-u.ac.jp)
SUMMARY
The past decade or so has seen rapid progress in our understanding of how left-right (LR) asymmetry is generated in vertebrate embryos. However, many important questions about this process remain unanswered. Although a leftward flow of extra-embryonic fluid in the node cavity (nodal flow) is likely to be the symmetry-breaking event, at least in the mouse embryo, it is not yet known how this flow functions or how the asymmetric signal generated in the node is transferred to the lateral plate. The final step in left-right patterning – translation of the asymmetric signal into morphology – is also little understood.
Introduction
There are two key steps that contribute to the early establishment of left-right (LR) patterning in the mouse. The first step is the symmetry-breaking event that takes place in the node around embryonic day (E) 7.5 of mouse development (Fig. 1). In this step, an asymmetric signal(s) that is generated in the node is transferred preferentially towards the left side of the lateral plate mesoderm (LPM). (This mesoderm is located in the lateral region of the early-somite-stage mouse embryo and later contributes to the mesenchyme of various visceral organs.) The transfer of this signal results in the second step: the asymmetric expression of the gene Nodal in the left LPM (see Fig. 1A,B). Cells in the left LPM that receive Nodal signaling contribute to various visceral organs, such as the lung and heart, that develop left side-specific morphologies.
In this review, we discuss our current understanding of the mechanism of
left-right (LR) patterning during development. In particular, we focus on
genetic data from the mouse; we do not discuss finding from studies in other
vertebrates, except where specifically mentioned. [For recent reviews of the
similarities and differences in LR patterning between the mouse and other
vertebrates, see Levin and Tabin (Levin,
2005
; Tabin,
2005).]
Leftward fluid flow breaks LR symmetry
Although there is some controversy concerning the initiation of LR
asymmetry in other vertebrates (see Tabin,
2005), at least in the mouse, the breaking of LR symmetry is most
likely to be achieved by the unidirectional flow of extra-embryonic fluid in
the node (the node is an embryonic structure that is located at the midline,
at the anterior tip of the primitive streak in mouse embryos, see
Fig. 1B and
Fig. 2A). This fluid flow is
referred to as nodal flow (Nonaka et al.,
1998). This leftward laminar flow of extra-embryonic fluid in the
node cavity occurs at a speed (visualized with fluorescent beads, see
Fig. 2D) of 
15 to 20
µm/second and is generated by the rotational movement of 9+0 monocilia
(these are cilia that have nine doublets of microtubules but that lack a pair
of central microtubles), which protrude from cells located on the ventral side
of the node into the node cavity (Sulik et
al., 1994) (Fig.
2C). These 200-300 cilia rotate in the same direction (clockwise,
as viewed from the ventral side) at a speed of 600 rpm
(Nonaka et al., 1998). Nodal
flow takes place for only a short period of time. It is, thus, first apparent
at the one- to two-somite stage, persists for several hours and ends by the
six-somite stage. The asymmetric expression of Nodal begins in the
LPM at the two-somite stage and disappears by the six-somite stage. Nodal flow
may therefore occur specifically to initiate Nodal expression on the
left side of the LPM. Monocilia are also present in the notochordal plate
(Nonaka et al., 1998), but
they are reported to be immotile (Okada et
al., 2005).
Nodal flow is essential for LR determination. Various mouse mutants that
lack normal flow as a result of the absence or immotility of node cilia are
reported to have abnormal LR patterning
(Okada et al., 1999). The
direct consequence of the absence of nodal flow appears to be the
randomization of LR orientation, as is best exemplified by the iv/iv
(inversus viscerum) mouse mutant, which possesses immotile cilia
(Supp et al., 1999;
Supp et al., 1997). However,
most mouse mutants that lack node cilia exhibit complex phenotypes (typically,
bilateral left sidedness, known as left isomerism) because they also have
functional defects in the midline barrier. The best examples of such mutants
are those that lack intraflagellar transport proteins, such as polaris (Ift88
– Mouse Genome Informatics), wimple (Wim; Ift172 – Mouse Genome
Informatics) and Kif3 proteins
(Garcia-Garcia et al., 2005;
Huangfu and Anderson, 2005;
Huangfu et al., 2003;
Murcia et al., 2000;
Nonaka et al., 1998).
Reversal of the direction of nodal flow by the imposition of an artificial
flow leads to the reversal of LR patterning in mice
(Nonaka et al., 2002),
demonstrating that the flow per se directs subsequent LR patterning events.
Recent evidence suggests that a similar mechanism may operate in other
vertebrates (Okada et al.,
2005). In zebrafish, for example, cilia have been detected in
Kupffer's vesicle, the embryonic organizer equivalent to the mouse node
(Essner et al., 2002;
Essner et al., 2005;
Kramer-Zucker et al., 2005).
These cilia are motile and generate a unidirectional flow in the vesicle.
Zebrafish mutants that lack the cilia in Kufpper's vesicle show impaired LR
patterning (Essner et al.,
2005; Kawakami et al.,
2005; Kramer-Zucker et al.,
2005), but the role of the flow has not been directly tested.
The origin of LR polarity
The mechanism of symmetry breaking must make use of the preexisting
positional cues: the anteroposterior (AP), dorsoventral (DV) and mediolateral
axes. As Brown and Wolpert proposed in their conceptual F-molecule model
(Brown et al., 1991), the
origin of the LR axis must derive from AP and DV axis information. But how is
AP and DV information translated into LR polarity? This question is now known
to be equivalent to: how is such information translated into the generation of
the leftward flow in the node? Furthermore, if all the node cilia rotate in
the same clockwise direction, how can they generate a unidirectional flow?
The key to the answer to this last question was recently shown to lie in
the rotation angle of the cilia (Okada et
al., 2005; Nonaka et al.,
2005). Hydrodynamic principles dictate that a simple rotational
movement of cilia would generate a vortex only if the cilia protrude
vertically from a surface. However, if the cilia are tilted towards a specific
direction, it is possible for them to generate a unidirectional flow
(Cartwright et al., 2004;
Nonaka et al., 2005;
Okada et al., 2005). Thus,
when a cilium moves closer to the surface, the movement of fluid near the
surface will be restricted as a result of the so-called no-slip boundary
effect [according to this effect, fluid contacting a solid wall will not move;
owing to its viscous force, this static fluid layer would prevent the fluid
that overlies it from being dragged by the cilia
(Liron, 1996)]. Conversely,
when a cilium moves away from the surface, it induces the movement of the
neighboring fluid more effectively. If cilia are tilted toward the posterior
side, they would be moving towards the right when they come close to the
surface and towards the left when they are far from the surface (see
Fig. 3). Hydrodynamics
therefore predict that a leftward flow can be generated if rotating cilia are
tilted towards the posterior side. High-speed microscopic observations have
revealed that the node cilia are indeed tilted posteriorly, at an average
angle of 30° (Nonaka et al.,
2005; Okada et al.,
2005).
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).
How does AP information dictate the positioning of each node cilium in a
posteriorly tilted manner? A careful examination of the node cilia
(Nonaka et al., 2005;
Okada et al., 2005) has
revealed that most of them protrude from the posterior region of node pit
cells (cells on the ventral surface of the node that have monocilia, see
Fig. 1C and
Fig. 3). The basal body of each
cilium is also positioned posteriorly within the cell
(Fig. 3). Node pit cells are
roughly spherical. As such, the protrusion of cilia perpendicular to the cell
membrane and the posteriorly shifted localization of the basal body may
explain the tilt of the cilia toward the posterior side. This scenario is
similar to the planar cell polarity (PCP) pathway that is responsible for the
coordinated localization and orientation of hairs in Drosophila
cuticles and of sensory hairs in the vertebrate inner ear
(Klein and Mlodzik, 2005).
Similarly, DV positional information may determine the positioning of the
cilia according to apicobasal polarity of the epithelium. It appears likely
that a mechanism similar to PCP operates to correctly position the basal body
of each cilium in the node pit cells.
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Nodal flow is the event that most probably breaks LR symmetry during development, at least in the mouse, but the mechanism by which it achieves this effect has been subject to debate. Several models have been proposed, but there are two principal hypotheses (Fig. 4).
Does nodal flow transport a determinant?
This is an obvious possibility that was initially proposed by Nonaka et al.
(Nonaka et al., 1998).
According to this model (Fig.
4A), a molecule that acts as the LR determinant is transported by
nodal flow towards the left side. If the molecule is secreted by node cells
(such as pit cells located in the node cavity or perinodal crown cells) into
the node cavity, it would be readily transported by the fluid flow. Indeed,
recent evidence (Tanaka et al.,
2005) indicates that node pit cells actively secrete vesicles,
called nodal vesicular parcels (NVPs), that appear to contain Hedgehog (Hh)
protein and retinoic acid (RA) into the node cavity. However, the identity of
such a transported molecular determinant of LR polarity remains to be
established, Furthermore, the identity of the cells on the left side that
receive the determinant signal is unknown. Several candidates have been
proposed for the putative LR determinant, such as sonic hedgehog
(Shh) and RA (Tanaka et al.,
2005), but none seems to fulfill all the required criteria.
According to these criteria, a candidate for the LR determinant should be
produced in or near the node, and its loss should result in the lack of
Nodal expression in the lateral plate.
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) but not in other vertebrates, including the mouse. Shh
is expressed in the midline, including the node, and
Shh–/– mice exhibit LR defects
(Meyers and Martin, 1999).
However, Shh–/– embryos show left isomerism as
a result of impaired midline function. The LR decision is initially normal in
the Shh-null embryos, but they subsequently develop bilateral
Nodal expression in the lateral plate because of the midline
abnormality (see below for a discussion of the function of the midline).
Another Hedgehog family protein, Indian hedgehog (Ihh), is also implicated in
LR patterning. Mice lacking both Shh and Ihh, similar to smoothened
(Smo)-null mice, fail to develop asymmetric Nodal expression
in the lateral plate (Zhang et al.,
2001). However, the LR defects of these animals are most likely to
be caused by the associated lack of Gdf1 expression, which is
required for asymmetric Nodal expression in the LPM
(Rankin et al., 2000).
Moreover, an examination of the expression of Hh target genes, such as
Ptch1, failed to reveal any asymmetry in Hh signaling in normal mouse
embryos (Zhang et al., 2001).
These observations suggest that, in the mouse, Hh signaling is required for
the formation of a functional midline, including the node, but that it is not
directly involved in LR determination.
RA is synthesized in regions near the node by the enzyme retinaldehyde
dehydrogenase 2 (RALDH2). RA signaling, as revealed by expression of an
RA-responsive transgene, has also been detected in the perinodal region
(Vermot et al., 2005).
However, mice that lack RALDH2, the only RA-synthesizing enzyme expressed near
the node, exhibit normal LR patterning in the lateral plate; that is,
asymmetric Nodal expression is maintained
(Vermot et al., 2005). It is,
thus, unlikely that RA, which was shown to be contained in NVPs
(Tanaka et al., 2005),
regulates the LR decision at the node. Instead, RA is required for the
maintenance of bilateral symmetry during somite formation
(Kawakami et al., 2005;
Vermot et al., 2005;
Vermot and Pourquie,
2005).
Fibroblast growth factor 8 (Fgf8) has also been considered as a candidate
for the LR determinant that is transported by nodal flow. Mice conditionally
deficient in Fgf8 lack Nodal expression in the LPM and
exhibit right isomerism (Meyers and
Martin, 1999). Superficially, Fgf8 appears to be required for
determination of the left side. Experiments with an Fgf inhibitor (SU5402)
suggest that Fgf8 may be necessary for the secretion of NVPs
(Tanaka et al., 2005), but it
remains uncertain exactly when and where Fgf8 plays a role in LR patterning.
Fgf8 may thus function in the node or in the LPM to maintain the competence of
the LPM to respond to the Nodal signal, for example. The site-specific
ablation of Fgf8 will be necessary to clarify the precise site of
Fgf8 function.
Does nodal flow generate mechanical stress?
An alternative to the hypothesis that nodal flow transports an LR
determinant is that it generates mechanical stress that is sensed by node
cells, either pit cells or crown cells
(Fig. 4B) [crown cells are
peri-nodal cells of endoderm origin that express Nodal, Gdf1 and
Cerl2 (Dand5 – Mouse Genome Informatics); see
Fig. 1B]. There are several
examples of cells that sense flow-generated mechanical stress (for a review,
see Orr et al., 2006), perhaps
the best known of which is the sensing of blood flow by vascular endothelial
cells. The magnitude of mechanical stress generated by a flow depends on
several factors, including the speed of the flow and the viscosity of the
fluid. As mentioned above, the speed of nodal flow, as visualized with
fluorescent beads, is 15 to 20 µm/second, which is much slower than
that of blood flow in peripheral arteries (10 mm/second). The precise
viscosity of the extra-embryonic fluid present in the node cavity is unknown,
but the viscosity of bovine amniotic fluid is 3 to 5 mPa·s, which is
similar to that of blood (14 mPa·s). The Reynold number (Re,
which is the ratio between inertial force and viscous force and is very low
for microscopic phenomena) for ciliary rotation is only
5x10–4
(Cartwright et al., 2004),
suggesting that the associated inertial force is negligible. Therefore, the
shear stress generated by nodal flow might be too small to be sensed by the
cell surface.
The mechanical stress experienced by a cell may also depend on which
subcellular organelle is responsible for its detection. Shear stress can be
detected by the cell membrane (as in vascular endothelial cells), as well as
potentially by nonmotile cilia (McGrath et
al., 2003). If the latter is the case, a small physical force
generated by the slow nodal flow might be amplified by the bending of the
cilia. Alternatively, if the mechanical stress is sensed by the membrane of
node cells, the signal may be amplified within the cell by signaling
cascades.
Recently, a family of mechanically gated channels called TRP channels has
been implicated in mechanotransduction in sensory systems, including hearing
and touch sensitivity (Pedersen et al.,
2005). Interestingly, some of the TRP family members contain
ankyrin repeats, which may serve as a molecular spring to amplify a small
level of shear stress (Lee et al.,
2006). It may be interesting to search for the expression of TRP
members in or near the node.
Role of a Ca2+ signal
Two lines of evidence indicate a role for Ca2+ in LR
determination downstream of nodal flow, which possibly favor the
mechanosensory model. First, asymmetric Ca2+ signaling has been
detected at the left margin of the node
(McGrath et al., 2003). The
asymmetric elevation of Ca2+ and its lateral propagation have also
been reported (Tanaka et al.,
2005). Second, a putative Ca2+ channel has been
implicated in LR decision making. PKD2, a causative gene for
autosomal recessive polycystic kidney disease in humans, encodes a protein
that functions as a Ca2+ release channel in cultured cells
(Luo et al., 2003).
Pkd2 mutant mice develop LR defects that are consistent with impaired
mechanosensation. They, thus, possess morphologically normal and motile cilia
but fail to develop asymmetric expression of Nodal in the LPM
(Pennekamp et al., 2002). They
also exhibit a low level of bilateral Pitx2 expression in the
posterior lateral plate (LP) [Pitx2 is a transcription factor, the expression
of which is induced in left LPM by Nodal signaling
(Shiratori et al., 2001)].
However, this is probably due to a decrease in the level of the signal that is
required to activate Nodal expression in the LPM, given that a
similar phenotype is apparent in conditional Foxh1 mutants
(Yamamoto et al., 2003) and
can be simulated by a theoretical model known as the reaction-diffusion system
(T. Nakamura, N. Mine, E. Nakaguchi, M. Yamamoto, K. Yahsiro, C. Meno and
H.H., unpublished). Foxh1 is a transcriptional factor that mediates Nodal
signaling (Hoodless et al.,
2001; Yamamoto et al.,
2001) and is required to upregulate Nodal and
Lefty2 expression in the left LPM
(Saijoh et al., 2000;
Yamamoto et al., 2003). Recent
observations support the notion that Pkd2 acts as a Ca2+ channel
that is gated by mechanical stress. First,
Pkd2–/– embryos do not manifest asymmetric
Ca2+ signaling in the perinodal endoderm
(McGrath et al., 2003).
Second, kidney epithelial cells derived from
Pkd2–/– embryos fail to respond to mechanical
flow, while wild-type ones do so (Nauli et
al., 2003; Nauli and Zhou,
2004). Although Pkd2 may be a component of a mechanosensor, the
precise role of Pkd2 in LR determination remains to be clarified. Given that
Pkd2 is expressed ubiquitously, Pkd2 may potentially function during
LR patterning at any site between, and including, the node and LPM. The
subcellular localization of Pkd2 has also been controversial, as it has been
detected in association with a variety of organelles, including the plasma
membrane, Golgi apparatus (Scheffers et
al., 2002), mitotic spindle
(Rundle et al., 2004) and
cilia (Yoder et al., 2002). If
it is localized to the surface of node cilia, Pkd2 may serve as a
mechanosensory, as proposed by the two-cilia model
(McGrath et al., 2003;
Tabin and Vogan, 2003), which
proposes that two kinds of node cilia exist in the node: motile cilia that are
positive for a dynein called Lrd (Supp et
al., 1997), which generate the flow; and immotile Lrd-negative
cilia, which presumably act as mechanosensors.
Hints from kidney cilia
Epithelial cells of the kidney collecting duct also possess 9+0 monocilia,
but, unlike the node cilia, they are immotile. Many of the genes that cause
polycystic kidney, including Pkd2, also play a role in LR patterning,
indicating that there may be a similarity in function between the cilia of the
kidney and those of the node. Polaris is a protein that contributes to
intraflagellar transport, and its deficiency results in polycystic kidney, as
well as in LR defects characterized by bilateral Nodal expression in
LPM and left isomerism (Murcia et al.,
2000). As mentioned above, inv is a rare mutation that
gives rise to situs inversus, but it also results in polycystic kidney in
homozygotes (Yokoyama et al.,
1993). The Inv protein localizes to both node cilia and kidney
cilia (Watanabe et al.,
2003).
Recent evidence suggests that renal cilia function as flow sensors, as
removal of the primary cilium from Madin-Darby canine kidney (MDCK) cells
abolishes flow sensing (Praetorius and
Spring, 2003). Mechanical stress also stimulates Ca2+
signaling in kidney epithelial cells. Mechanical bending of the cilium of MDCK
cells either with a micropipette or by artificial flow induces an increase in
the intracellular Ca2+ concentration
(Praetorius and Spring, 2001).
Kidney epithelial cells in primary culture also manifest intracellular
Ca2+ signaling in response to artificial flow. However, such cells
deficient in Pkd2 fail to respond to flow
(Nauli et al., 2003).
Role of Invs
The inv mouse mutation induces situs inversus (a malformation in
which the LR asymmetry of the viscera is completely reversed), instead of LR
randomization, in virtually all homozygotes
(Yokoyama et al., 1993). Nodal
flow in inv mutant mice is slow and turbulent, but its direction is
still leftwards (Okada et al.,
1999). A leftward artificial flow is able to correct situs
inversus in such mutant embryos (Nonaka et
al., 1998; Watanabe et al.,
2003), suggesting that the endogenous flow is abnormal. However,
examination of the node cilia of inv/inv embryos has failed to reveal
obvious anomalies in their structure or movement
(Watanabe et al., 2003), but a
recent report (Okada et al.,
2005) showed that a small proportion of the node cilia are
abnormally tilted. The Invs gene encodes a protein that contains
ankyrin repeats (Mochizuki et al.,
1998; Morgan et al.,
1998) and is preferentially localized to 9+0 cilia, including
those of the node (Watanabe et al.,
2003). Invs is expressed ubiquitously, however, and the
encoded protein is present in both the cytoplasm and nucleus. Biochemical
assays have shown that the Invs (inversin) protein interacts with a variety of
other proteins, including calmodulin
(Yasuhiko et al., 2001),
dishevelled 1 (Dvl1) (Simons et al.,
2005), ß-cadherin and N-cadherin
(Nurnberger et al., 2002), and
a component of the anaphase-promoting complex
(Morgan et al., 2002). The
precise function of Invs remains unknown.
Chemosensory versus mechanosensory models
Additional observations may be relevant to the role of nodal flow. It has
been thought that data obtained with artificial flows are inconsistent with
the transport model because such fast flows would be expected to disperse a
soluble determinant molecule. However, this conclusion might be incorrect.
Although a fast flow was imposed on embryos in the study by Nonaka et al., the
effective flow speed in the node cavity (20 µm/second) was within the range
of physiological flows (Nonaka et al.,
2002). Therefore, depending on which cells receive a determinant
molecule, these data do not completely exclude the transport model. It has
also been argued that the phenotypic difference between mutants with immotile
cilia and those lacking cilia favors the mechanosensory model. Thus, whereas
iv mutant mice, which possess immotile node cilia, show randomized
Nodal expression in the LPM, embryos without node cilia (such as
those deficient in Kif3a, Kif3b, polaris and wimple) exhibit bilateral
Nodal expression (Nonaka et al.,
1998; Murcia et al.,
2000; Huangfu and Anderson,
2005). However, such a difference in Nodal expression
might simply be due to the presence or absence of midline defects. The
iv mutant does not have a midline defect, whereas the latter mutants
all do (they all lack Lefty1 expression). In our view, the difference
in phenotype between these two types of mutant does not support one model over
the other.
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Signal transfer from the node to the lateral plate
The asymmetric signal (or signals) generated in or near the node, whether it is mechanical stress or a molecular determinant, must be transferred to the lateral plate, where it induces the asymmetric expression of Nodal (Fig. 1, Fig. 5). Several important questions remain unanswered about this process. How and through which route is the signal transferred to the LPM? How does the signal activate Nodal expression in the left LPM? What is the nature of the signal that reaches the left LPM and activates Nodal expression there? Asymmetric elevation of Ca2+ may be an intermediate event between the node and the LPM, but how is it related to the asymmetric Nodal expression in LPM? These questions do not address separate issues, but rather are interrelated.
Route of signal transfer
Whether the cilia-derived asymmetric signal is a molecule or mechanical
stress, the topology of the mouse embryo is an important determinant of its
transfer. Several potential routes can be envisioned for the transfer of a
signal generated in or near the node to the LPM
(Fig. 1C). This route may
include: the node pit cells with their rotating cilia; node cells with
immotile cilia, as suggested by the two-cilia model
(McGrath et al., 2003;
Tabin and Vogan, 2003); crown
cells in the perinodal region; or endoderm cells distantly located from the
node.
Is the signal from the node to the LPM relayed or directly transferred?
How is the LR signal transferred from the node to the lateral plate?
Signaling molecules expressed in the node are essential for correct LR
patterning of the lateral plate, and they may play a role in the transfer of
the LR signal. Nodal is bilaterally expressed in the node (in
perinodal crown cells) before its expression begins in the left LPM (see
Fig. 1C). Furthermore, genetic
evidence has established that Nodal expression in the node is
essential for subsequent Nodal expression in the left LPM
(Brennan et al., 2002;
Saijoh et al., 2003). The
specific ablation of Nodal expression in the perinodal region has
been shown to prevent Nodal expression in the left LPM
(Brennan et al., 2002).
Cerl2, which encodes an antagonist of Nodal, is also expressed in the
perinodal region before Nodal expression begins in the left LPM
(Marques et al., 2004).
Cerl2 is expressed in an asymmetric manner, with the level of
expression on the right side being substantially higher than that on the left
side (Pearce et al., 1999).
Mice that lack Cerl2 show bilateral or right-sided expression of
Nodal in the LPM (Marques et al.,
2004), suggesting that this Nodal antagonist produced in the node
regulates the asymmetric expression of Nodal in the LPM. These
observations thus indicate that Nodal may play a role in signal transfer from
the node to left LPM.
Nodal is currently the only signaling molecule whose function in the node
has been established by genetic means to be essential for Nodal
expression in the LPM (Brennan et al.,
2002; Saijoh et al.,
2003). Any model for LR patterning must therefore take this fact
into consideration. But, what is the precise role of Nodal produced in the
node? The Nodal signal might be relayed to the LPM. Nodal produced in the node
may thus act on cells that are located between the node and the LPM (such as
perinodal cells, endodermal cells distantly located from the node, paraxial
mesoderm cells or intermediate mesoderm cells) and induce in them a secondary
signal that travels to the LPM and activates Nodal expression. In the
chick embryo (Rodriguez Esteban et al.,
1999; Yokouchi et al.,
1999), Shh produced in the node activates the expression of
Caronte in the paraxial mesoderm, which encodes an inhibitor of bone
morphogenetic protein (BMP). Caronte, in turn, induces Nodal
expression in the left LPM. However, it is not known whether a similar
signaling mechanism operates in other vertebrates. A BMP antagonist
corresponding to Caronte has not been identified in the mouse or zebrafish
genomes. Nevertheless, a BMP signal may negatively regulate Nodal
expression in the LPM of the mouse embryo, given that, in the absence of the
BMP effectors Smad1 and Smad5 (Chang et
al., 2000), Nodal is expressed bilaterally in the
LPM.
An alternative is that Nodal itself is transported from the node to the
left LPM. Several lines of circumstantial evidence support this possibility.
First, Nodal expression in the LPM can be induced by Nodal itself.
Thus, the ectopic introduction of a Nodal expression vector in the right LPM
induces the expression of endogenous Nodal
(Yamamoto et al., 2003).
Second, a search for transcriptional regulatory sequences that control
asymmetric Nodal expression has identified two enhancers, both of
which are able to confer asymmetric gene expression in the left LPM
(Adachi et al., 1999;
Norris and Robertson, 1999;
Saijoh et al., 2000).
Importantly, both enhancers possess binding sequences for the transcription
factor Foxh1 that are essential for enhancer activity and Nodal responsive
(Saijoh et al., 2005).
However, the transport of Nodal from the node to the left LPM remains to be
directly demonstrated.
GDF1 is a transforming growth factor ß (TGF-ß)-related protein
that is expressed in the node and that plays a role in LR patterning. Like
Nodal, GDF1, which shares sequence similarity with Vg1 in Xenopus, is
bilaterally expressed in the perinodal region. Mice that lack GDF1 do not
manifest asymmetric Nodal expression in the LPM and exhibit right
isomerism of the visceral organs (Rankin
et al., 2000). Similarities in Gdf1 and Nodal
expression domains and their respective mutant phenotypes indicate that GDF1
may play a role in transferring a laterality signal from the node to the LPM
by interacting with Nodal. However, evidence suggests that GDF1 may also play
a different role in LR patterning. First, Gdf1 is expressed not only
in the perinodal region but also in the LPM at the early somite stages,
indicating that GDF1 renders the LPM competent to respond to the Nodal signal.
Second, GDF1 alone can activate signaling by Nodal signaling components, as
well as by a Nodal responsive reporter when overexpressed in frog embryos or
cultured cells (Cheng et al.,
2003; Wall et al.,
2000), suggesting that GDF1 may play a role in LR patterning
independently of Nodal.
It thus remains unknown how the LR signal travels from the node to the lateral plate. Clarification of this issue will require us to understand the precise role of Nodal (and of GDF1) produced in the node.
Lateral plate asymmetric patterning by positive and negative signaling loops
The asymmetric patterning of the lateral plate is perhaps the best
understood step of the establishment of the LR axis. Nodal, Lefty1 and Lefty2
play a central role in this process
(Capdevila et al., 2000;
Hamada et al., 2002). Genetic
evidence has revealed that Nodal acts as a left-side determinant. Cells in the
left LPM that have received the Nodal signal contribute to the left
side-specific morphology of various visceral organs (as discussed in more
detail below), whereas cells in the right LPM, which do not receive the Nodal
signal, contribute to right side-specific morphology. Thus, in the absence of
Nodal signaling, bilaterally asymmetric visceral organs, such as the lungs,
adopt right isomerism.
Lefty1 is a feedback inhibitor of Nodal that restricts the area of Nodal
signaling and the duration of Nodal expression. Mammals possess two
Lefty genes, Lefty1 and Lefty2, which are expressed in the
midline and left LPM, respectively (Fig.
5). Their expression is induced by Nodal signaling
(Yamamoto et al., 2003). In
the absence of Lefty1 or Lefty2, asymmetric Nodal expression in the
LPM begins normally, but the Nodal signal subsequently leaks to the right
side, resulting in bilateral Nodal expression
(Meno et al., 1998;
Meno et al., 2001).
The expression of Nodal and Lefty genes is dynamic and transient.
In the mouse embryo, Nodal expression in the LPM begins in a small
region on the left at the level of the node and subsequently expands within
the left LPM along the AP axis (Fig.
6). Nodal produced in left LPM induces Lefty2 expression
in the left LPM (Fig. 5). Nodal
is also thought to travel to the midline, where it induces Lefty1
expression (Yamamoto et al.,
2003). Expression of Nodal (as well as that of
Lefty1 and Lefty2) then starts to decrease and has
completely ceased by the six-somite stage. Asymmetric expression of
Nodal in LPM thus persists for only 6 hours. Such dynamic
expression of Nodal and Lefty genes is achieved by positive and
negative regulatory loops that are mediated by Nodal and the Lefty proteins.
Thus, the expression of both Nodal and Lefty2 is regulated
by the Nodal responsive enhancer ASE
(Adachi et al., 1999;
Norris and Robertson, 1999;
Saijoh et al., 1999;
Saijoh et al., 2000). This
system ensures the presence of the Nodal signal at the correct time and
place.
|
;
Hamada et al., 2002;
Juan and Hamada, 2001;
Saijoh et al., 2000;
Branford and Yost, 2004) have
suggested that these secreted factors constitute a reaction-diffusion system,
a theoretical model that is able to convert a small difference between two
separated regions into a robust difference through local activation and
long-range inhibition (Turing,
1990; Meinhardt and Gierer,
2000). This notion is further supported by: the dynamic expression
patterns of Nodal and both Lefty genes; the ability of Nodal and both
Lefty proteins to act over a long distance; and the construction of a
mathematical model that can simulate in vivo data for wild-type and various
mutant embryos (T. Nakamura, N. Mine, E. Nakaguchi, M. Yamamoto, K. Yahsiro,
C. Meno and H.H., unpublished). It is, thus, possible that nodal flow
generates only a small difference, which is converted to robust asymmetry by a
reaction-diffusion system (Fig.
7).
Despite such progress, important questions related to asymmetric patterning
in the LPM remain unanswered. First, LPM on both sides is connected at the
posterior end. Given that the introduction of a Nodal expression vector into
the right LPM activates expression of endogenous Nodal
(Yamamoto et al., 2003), left
LPM and right LPM are equally competent to respond to the Nodal signal. What
then is the mechanism that prevents the Nodal signal from leaking across the
midline at the posterior end? In the absence of Lefty2 in the LPM, the Nodal
signal (as revealed by the expression of Pitx2) extends to the
posterior region of right LPM (Meno et
al., 2001). Is the inhibitor Lefty2 sufficient to prevent leakage
of the Nodal signal, or is there an additional mechanism that restricts Nodal
signaling? The autonomous positive-negative regulatory loops mediated by Nodal
and Lefty proteins may operate to reduce the level of Nodal
expression as it approaches the posterior midline, but such a mechanism would
not be expected to be error free.
|
).
Theoretical simulation (Meinhardt and
Gierer, 2000) also suggests that a diffusible inhibitor produced
at the midline (Lefty proteins) establishes asymmetric Nodal
expression in the LPM by negatively regulating Nodal over a long
distance (Fig. 5B, step 4).
However, is Lefty1 alone sufficient for barrier function? What is the nature
of the midline barrier in the posterior region where Lefty1
expression is absent? The mechanism that restricts the Nodal signal
exclusively to the left side is thus not fully understood. Situs-specific organogenesis: the final step of LR signal interpretation
Less clearly understood is the final step of LR patterning, situs-specific organogenesis. It is not currently known how LR asymmetric information is conveyed to give rise to asymmetric organogenesis. Anatomic asymmetries become recognizable in various visceral organs only after asymmetric Nodal expression in the LPM has disappeared. Macroscopically, at least three different mechanisms are responsible for generating asymmetric structures (Fig. 8). The first is directional looping: organs that are initially formed as a tube, including the heart and gut, undergo a series of looping, bending and rotation steps that result in their correct positioning within the body (Fig. 8A). In the second, a pair of primordial organs that form symmetrically on both sides subsequently develop differences in their size or branching pattern, as is the case for the lungs (Fig. 8B). And in the third, one side of a symmetric structure, such as a blood vessel, undergoes regression and disappears, leaving only the other side (Fig. 8C).
|
). LR
signaling may induce differential cell death in developing arteries, but how
the LR signal is interpreted by the arteries is unknown. By contrast,
directional looping of the gut in the chick embryo is thought to be mediated
by differential cell elongation (Muller et
al., 2003). Looping of the zebrafish gut, however, results from
the asymmetric migration of left and right LPM
(Horne-Badovinac et al.,
2003). The left LPM thus migrates dorsally to the endoderm, while
the right LPM migrates ventrolaterally, resulting in a shift of the developing
intestine to the left. Genetic evidence indicates that asymmetric LPM
migration is dependent on LR signaling
(Horne-Badovinac et al.,
2003), but it remains unknown how the direction of cell migration
is regulated. Cardiac looping, the first visible indication of asymmetric
morphogenesis, has received much attention because impaired looping results in
congenital heart malformations in humans. However, the cellular and
biophysical bases of cardiac looping remain unknown. The looping may be
achieved by forces intrinsic to the heart tube, such as those generated by
changes in myocardial cell shape (Manasek
et al., 1972) or in the arrangement of intracellular actin bundles
(Itasaki et al., 1991;
Itasaki et al., 1989).
Alternatively, the looping may be driven by external forces, such as that
exerted by the splanchnopleure (Voronov et
al., 2004). The splanchnopleure is the rudiment of the spleen that
forms as a single condensation of mesenchyme along the left side of the
mesogastrium dorsal to the stomach. The formation of this rudiment, which
comprises a group of cells positive for the transcription factors Hox11 and
Nkx2.5, depends on a columnar mesoderm-derived cell layer known as the
splanchnic mesodermal plate (SMP)
(Hecksher-Sorensen et al.,
2004). The SMP is bilateral at early stages, surrounding the
prospective stomach located at the midline. Under the control of the LR
signal, however, the left SMP grows laterally while the right SMP disappears.
Differential cell proliferation is at least one of the mechanisms responsible
for the asymmetry in the SMP
(Hecksher-Sorensen et al.,
2004). The SMP may induce the formation of both the splenic
mesenchyme and the pancreas bud.
At the molecular level, the main player that regulates asymmetric
organogenesis downstream of the Nodal signal is the transcription factor Pitx2
(Logan et al., 1998;
Yoshioka et al., 1998). Like
Nodal and Lefty2, Pitx2 is expressed asymmetrically in left
LPM, but asymmetric expression of Pitx2 persists until much later
stages than does that of these other two genes. An analysis of transcriptional
regulatory elements has indicated that asymmetric Pitx2 expression is
induced by Nodal and is maintained by Nkx2 in the absence of the Nodal signal
(Shiratori et al., 2001). Mice
that lack Pitx2 (specifically, Pitx2c, the isoform that is asymmetrically
expressed) exhibit laterality defects in most visceral organs
(Liu et al., 2001).
LPM-derived cells that express Pitx2 activity develop left-side morphologies.
Thus, in the absence of Pitx2, bilateral organs, such as the lungs, exhibit
right isomerism. However, certain laterality events, such as cardiac looping
and embryonic turning, take place normally in the absence of Pitx2, suggesting
that both Pitx2-dependent and -independent mechanisms are operative. How can
Pitx2 induce seemingly different cellular events, such as increased or
decreased cell proliferation, cell death and cell migration? It may regulate
distinct sets of genes in different organs, so that the readout of Pitx2
activity also differs. Currently, no target gene of Pitx2 that is relevant to
asymmetric morphogenesis has been identified. Pitx2 stimulates cell
proliferation in the pituitary gland by regulating cyclin genes
(Kioussi et al., 2002).
However, it is not known whether a similar mechanism operates in LPM-derived
cells.
Perspectives
The field on LR asymmetry has developed rapidly since the discovery of LR
asymmetrically expressed genes, such as Shh and Nodal in the
chick (Levin et al., 1995),
and Nodal and Lefty1 in the mouse
(Collignon et al., 1996;
Lowe et al., 1996;
Meno et al., 1996). We now
know that a large numbers of genes are involved in generating LR asymmetric
organs. The systematic screening of LR mutants will further identify those
genes that are required for normal LR patterning. Although LR asymmetry is an
interesting scientific problem to solve, it has a practical application as
well, as it is likely that many human congenital cardiac malformations are due
to defective LR patterning. The knowledge obtained from animal models will
therefore certainly contribute to our understanding of such abnormalities.
As is clear from this review, LR asymmetry is still a challenging topic! Many key questions remain to be answered despite recent progress. Key challenges in the immediate future are to clarify how nodal flow works, to know the nature of the asymmetric signal generated in the node and how it is transferred to the LPM, and to understand the cellular basis of asymmetric morphogenesis. To address these outstanding issues, it may be necessary to develop and integrate new techniques including imaging, in vitro culture and time-lapse observation systems, theoretical modeling, and more sophisticated genetic manipulations. In addition, there are many more issues that are taken for granted but that need to be examined in the near future (such as the exact role of the midline; the behaviors of secreted Nodal and Lefty proteins; and how and where BMP signaling acts during LR patterning). They may look less challenging but are certainly important issues. With steady progress and a breakthrough or two, we should be able to understand the whole process of LR asymmetry in the near future.
ACKNOWLEDGMENTS
We thank members of the Hamada laboratory for proposing various ideas, for discussions and for providing illustrations, and we thank unknown reviewers for their critical yet constructive comments. We apologize colleagues whose work we were unable to cite because of length constraint. The work performed in the authors' laboratory has been supported by CREST.
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