How genes establish left-right asymmetry in vertebrate development Word Count 1941

Introduction

While the vertebrate body exhibits bilateral symmetry externally, a remarkable conservation of asymmetry is displayed by the internal organs along the left-right body axis. This asymmetry manifested appears to be conserved in all vertebrates, implying that this asymmetric structure and arrangement of organs is vital and required for their normal function. In fact, asymmetric organ structures are beneficial in many ways. For instance, cardiac left-right asymmetry might more efficient as two separate pumping systems within a single structure are generated which allowed the increased functional complexity associated with the vasculature in vertebrates. Besides, the left-right asymmetries might also help in packing the organ systems, especially the digestive system, efficiently while maintaining the overall size limitations of an organism body cavity. Hence, the normal disposition of organs, called situs solitus, is an essential and distinctive feature of the vertebrate body plan.

Failure to establish a normal organ systems arrangement may result in distinct classes of laterality defects, namely isomerism (failure to achieve left-right asymmetry at the level of individual organs), heterotaxia (one or more of the individual organ systems develops with reserved left-right polarity), and situs inversus (a complete inversion of the global left-right axis). Although situs inversus is not associated with clinically detectable adverse effect, heterotaxia and isomerism are associated with a wide range of cardiopulmonary defects. In various genetic studies, four stages of left-right asymmetrical development were exhibited. The first stage the breaking of symmetry, then followed by the transfer of left-right positional information to the lateral plate mesoderm and stabilization of the side-specific gene expression and lastly the development of the asymmetric organs.

Breaking symmetry

The first stage in developing left-right asymmetry is about the breaking of early bilateral symmetry of the embryo such that the left-right axis becomes consistently oriented. This is achieved by integrating information concerning the relative orientations of the other

two primary exes of the embryo, the anteroposterior and dorsoventral axes. While there are several theoretical models proposed to explain this event, there is no apparent evidence to support these models.

The first model proposed was based on the discovery of monocilia on the ventral surface of the mouse node (the mammalian equivalent of the early embryonic organizer region). These monocilia projected into the extraembryonic space surrounding the egg cylinder, and demonstrated a novel type of vertical motion that generated a remarkable leftward flow of the extraembryo fluid in the node region (Okada et al., 1999). The nodal flow causes a difference in the relative distribution of extracellular inducers around the node, thus forming the left-right axis. Later, these extracellular inducers trigger respective activation of distanct signalling pathways on the left and right sides of the embryo (Supp & Bruechner, Molecular motors: the driving force behind mammalian left-right development, 2000). Intriguingly, this model is completely consistent with the obvious correlation between situs abnormalities and ciliary dysfunction in human observed by Afzelius (1976).

This model is strongly supported by the research done in iversus viscerum (iv) mutant mouse. A random incidence of either situs solitus or situs inversus in these mouse were observed. iv gene encodes for left-right dynein (Lrd) protein which is expressed in the ventral node cells and other cell types in the embryo (Supp, et al., 1999). In the studies done by Okada et al. (1999) and Supp et al. (1999), mice with defeated Lrd develops immotile nodal cilia and failed to produce any nodal flow. Apart from that, mice with deficiency of KIF3A and KIF3B (kinesin molecules required for assembly of nodal cilia) also exhibited complex situs defects (Marszalek et al., 1999; Nonaka et al., 1998). This suggest that nodal cilia does play a vital role in determining the left-right development of mice, and perhaps other vertebrates.

In the studies of chich embryo, a number of striking asymmetry were detected near the chick organizer (Hensens node) well before any overt signs of asymmetry can be detected in any other part of the embryo (Hamburger & Hamilton, 1951). Soon after that, a number of genes expressed asymmetrically at the chick node, including Sonic hedgehog (Shh) on the left and Fgf-8 on the right were observed (Boettger et al., 1999; Levin et al., 1995). This observation shows that these early asymmetries in chick are all centered at the node which suggests that a similar mechanism in breaking the bilateral symmetry might be shared

among vertebrate. Despite all the strong evidence supporting the nodal cilia hypothesis, such mechanism is still not found in chick left-right development.

Transfer of left-right positional information to the lateral plate mesoderm

After the small, stable domains of asymmetric gene expression are established in the node and perinodal area, these initial local asymmetries are expanded into much broader domains of side-specific gene expression that subsequently coordinate the asymmetric development of the various organs primordial. An apparent gene involved in this stage is the Nodal which is expressed in a broad domain at the left lateral plate mesoderm (LPM) in all vertebrate to date (Levin et al., 1995). According to Levin, laterality defects were observed in organisms with aberrant patterns of Nodal expression. Further, randomisation of situs determination was also observed in organism with Nodal misexpressed on the right LPM. This elucidates the significant role played by Nodal in coordinating the development of the global left-right axis.

Left-right axis determination in vertebrate is not merely dependent on Nodal, in fact, a combination of genes were involved to ensure the right left-right development can be achieved. In chick, Shh is known to induce Nodal expression in the left LPM (Levin et al., 1995). Meanwhile, the induction process is not direct where one or more secondary signals produced by cells in the intervening paraxial mesoderm are known to mediate the induction process (Pagan-Westphal & Tabin, 1998).

One of the mediating molecules discovered recently is the product of Cerberusrelated gene which is Caronte (Car) (Rodriguez-Esteban et al., 1999). The protein produced by Car gene binds to the TGF- -related ligands known as Bone Morphogenetic Proteins (BMPs) on the left LPM, preventing them from interacting with their receptors (Hsu et al., 1998). Car is expressed throughout the entire left LMP, consistent with its putative role as a key intermediate signal between Shh and Nodal. In addition, misexpression of Car on the right side induces Nodal ectopically in the right LPM (Hsu et al., 1998). According to Hsu et al. (1998), applying exessive BMP protein to the left LPM blocks the endogenous induction of Nodal. This indicates that BMP signalling functions to repress Nodal expression. While BMP gene expressed bilaterally in the LPM, expression of Car gene in the left LPM, in turn,

repress the activity of BMP via antagonizing an endogenous, thus induces the expression of Nodal only in the left LPM.

There are also evidence suggests that Nodal is involved in inducing and maintain its own expression within the left LPM, operating via a classical positive feedback loop. This hypothesis is based on the observation that mice deficient for the EGF-CFC gene Cryptic, which encodes cofactor for Nodal, fail to express Nodal in the left LPM, although the earlier asymmetric expression of Nodal in the perinodal region develops normally in these mice (Schier & Shen, 2000). This hypothesis is further supported by Saijoh et al. (2000) findings that Nodal can activate transcription from its own promoter in a mechanism that depends on the activities of EGF-CFCs and the transcription factor FAST2.

Stabilization of the sidespecific gene expression As the broad domains of asymmetric gene expression within the LPM established, the activated respective genes must remain confined to their original sides as failure to maintain distinct domain of side-specific gene expression can result in a wide range of laterality defects. In vertebrates, many genes were identified to regulate the stabilization of the sidespecific gene expression.

In chick, two genes, namely Fgf-4 and Fgf-8 were indentified to express exclusively on the right side of the node, in a pattern complementary to Shh (Boettger et al., 1999). According to (Rodriguez-Esteban et al., 1999), FGF protein, when applied to the left LPM, can inhibit the expression of normal left-sided genes including Car and Nodal. This observation suggest that FGF functions to prevent any activation of Shh-dependent, left sided pathway in cells to the right of the node. Thus, ensuring the fidelity of the initial left-right decision is maintained.

Another important gene which is expressed at the left LPM of mice is lefty-1. In the experiment done my Meno et al., (1998), inactivation of lefty-1 in mice results in ectopic expression of left-specific genes in the right LPM. It was proposed that lefty-1 protein might function at the midline by binding to Car, the presumed long-range signal that relays left0right information from the node to the left LPM, preventing Car from interfering with the BMP-mediated repression of nodal on the right side (Rodriguez-Esteban et al., 1999).

One additional mechanism that appear to restrict the range of Nodal signaling is the negative feedback role of lefty-2 gene expressed in the left LPM (Meno, et al., 1998). This gene is antagonistic to Nodal signaling by Lefty proteins as direct competition between Nodal and Lefty proteins for common receptor bindingn sites was involved (Cheng et al., 2000). This result in a fine-tuning of the amount of Nodal signal effectively received by cells (Schier & Shen, 2000).

Asymmetric organ development

All the mechanisms previously mentioned provide a specific consistent bias to the organogenesis process that will then results in then normal situs of organs. To ensure the asymmetric morphogenesis of organs, the left-right positional information encoded by Nodal must be received by cells in respective LPM and transduced to the nucleus, where it can effect stable changes in gene expression.

Pitx2 gene was found to be important factor acting downstream of Nodal in the left LPM (Schweickert et al., 2000). Pix2 expression found at the subsequent stages in the left LPM of several organ primordia which includes heart, gut and stomach. Schweickert et al., (2000) also found that Pitx2 induces its own transcriptions and acts as a maintenance mechanism after Nodal expression fades in the left LPM. It is believe that this molecule might be function to mediate the transfer of left-right information from the LPM to the developing organs. In the study of Pix2-deficient mice, done by Gage et al. (1999), Pitx2 defeciency can result in laterailty defects including right pulmonary isomerism. This finding is consisten with the putative role for Pitx2 as a left determinant.

Apart from Pitx2, zinc finger gene SnR and homeobox gene Nkx3.2 might also involved in directing various aspects of asymmetric organ development. According to Rodriguez-Esteban et al., (1999), SnR is normally express in the right LPM while Nkx3.2 in the left. In chick, Nodal repressed SnR activity while activate Nkx3.2 activity (Patel et al., 1999). Apart from that, Petal et al. (1999) also discovered that treatment of embryos with antisense oligonucleotides specific for SnR results in randomization of left-right development, accompanied by ectopic expression of Pitx2 in the right LPM. This observation suggests that

SnR functions as a repressor of Pitx2, and that Nodal activates Pitx2 in the left LPM through repression of SnR.

Figure 1. Summary of inductive interactions in the leftright developmental pathway (Ramsdell & Yost, 1998).

Conclusion

Left-right asymmetrical development in vertebrate involved a pool of genes working together. There are still many undiscovered genes that might be involved in this fascinating mechanism. While the statement that overall mechanism of left-right patterning are conserved

among vertebrates appears to be true, it is still not clear that to what degree the initial steps of left-right determination are shared among the different vertebrate classes.

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