Left-right axis formation is a very crucial developmental stage that takes place very early on in the developing embryo. There have been a vast number of studies conducted over the past 15 years in order to unravel the mechanisms behind this complex process. These studies have yielded some landmark findings as well as launching many new areas of interest that previously did not exist.
Superficially, vertebrates display left-right symmetry in their body shape. However, it is on the inside where left-right asymmetry of internal organs becomes very evident. Examples of this are the leftward displacement of the heart, pancreas, spleen and stomach and the rightward displacement of most of the liver. Left-right symmetry also influences paired organs on either side of the body. An example is how the left lung has two lobes and right lung has three lobes differ.
The above figure shows four variations in left-right asymmetry. Situs solitus is the normal arrangement of internal organs. Right and left isomerisms occur when organs are mirror imaged across the midline either from the left or right side. Situs inversus is a complete transposition of organs from left to right. Another defect known as Situs ambiguous (heterotaxia) also occurs where organs are scattered abnormally around the chest and abdomen. Except for situs inversus totalis defects in the left-right organ positioning leads to severe consequences1.
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Vertebrate left-right symmetry studies have been conducted in mice, zebrafish, chick and xenopus laevis embryo models with great effect. Before left-right (LR) axis patterning is initiated in the embryo it is preceded by the anterior-posterior (AP) and dorsal-ventral (DV) axis patterning. At this stage the embryo is symmetrically identical across the midline and awaits the initial symmetry breaking stage that will begin to induce LR asymmetry via downstream processes .
A lot of work has been put into determining the initial symmetry breaking stages in frog, chick and zebrafish embryos. In spite of this, no clear cut explanation or conserved mechanism has been provided in to the initial symmetry breaking stage in any of these species2. However, in the mouse embryo a LR symmetry breaking stage known as nodal flow has been proposed.
Nodal flow takes place in the ventral node which is a midline structure in the mouse embryo. Collectively monocilia located on each cell of the ventral node3 rotate in order to drive extra embryonic fluid containing a material to the left of the node. This leftward flow of material is what is known as nodal flow. This leads to an unbalanced accumulation of the material to the left of the node which further sets off a series of downstream processes. Evidence of nodal flow has been derived from mouse mutant models. A study conducted by Nonanka et al3 showed that mutations in the dynein motor components KIF3A and KIF3B inhibit the formation of the cilia and lead to LR axis defects.
The above figure shows the monocilia and their direction of motion as they drive the material on to the left hand side of the ventral node.
There have been many candidates for the identity of the material/molecule that the nodal cilia drive across including; Nodal, Growth and differentiation factor 1(Gdf1), Fibroblast growth factor 8 (FGF-8), the Hedgehog (HH)-like signal Sonic Hedgehog (SHH) and Retinoic acid (RA) 5. Recent studies carried out showed the active secretion of vesicle called nodal vesicular parcels (NVPs) by the node pit cells in to the cavity6. The NVPs appear to contain Hedgehog and Retinoic acid6. However, none of these molecules fulfil all the criteria which require the molecule to be formed at the node and its subsequent loss results in the loss of Nodal at the lateral plate mesoderm7. More importantly, the cells on the left of the node that receive the signal are as of yet unknown7.
As the above figure shows another postulated possible mechanism of the nodal cilia is the generation of mechanical stress. The 'two cilia'8 model suggests a mechanosensing model that involves the nodal flow creating increased fluid pressure on the left hand side8. This increase in pressure is subsequently sensed by fixed sensory monocilia. This results in the formation of an 'assymetric Ca2+ mediated transduction event'5.
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Overall, some experimental data supports the chemosensory model above the mechanosensory model and vice versa. As of yet there hasn't been any decisive evidence fully supporting either one. Also this model has only been shown to exist in the mouse model5. Therefore, it is unlikely that nodal flow is the common pathway in the initiation of the LR axis formation5.
A L-R coordinator?
Twin studies in chick suggest that there is no prepattern for L-R asymmetry before streak formation. When streaks are oriented 180Â° away from each other, each twin develops a properly oriented L-R axis independent of the twin axis. This argues against a prepattern and suggests that if the twin axes are well separated, each can establish its own L-R axis properly. The mouse nodalflowhypothesis also argues against a prepattern. In Xenopus, however, experiments suggest that the L-R axes may be established as early as the one cell stage (Fig. 3). UV treatment of Xenopus embryos at this stage disrupts microtubules and prevents cortical rotation. Such embryos can be rescued by tilting of the embryos, re-establishing the D-V and A-P axes, but the L-R axis is now randomized with respect to heart looping and gut coiling (Yost 1995). This suggests a prepattern in the Xenopus embryo that can be disrupted by loss of microtubules. The existence of a L-R coordinator peripheral to midline structures has also been inferred by the ability of an active form of Vg1, a TGFb family member, to completely invert the axis when injected in specific blastomeres on the right (Hyatt and Yost 1998). This suggests that Vg1 normally acts on the left, presumably in descendants of the L3 blastomere, to orient the L-R axis with the D-V and A-P axes. The L3 blastomere gives rise to structures outside of the axial mesoderm, including LPM, suggesting the coordinator acts outside of the midline (Dale and Slack 1987; Moody 1987). The ability of Vg1 to invert the axis cannot be mimicked by other TGFb members tested, suggesting a specificity for Vg1 in this process. However, many TGFb members converge upon common downstream effectors such as Smad2 and thus could functionally substitute for one another in such injection experiments (Gritsman et al. 1999). Since no active or processed Vg1 protein has been detected in Xenopus embryos, Vg1 may be mimicking the effect of another related TGFb family member that has not yet been tested in this assay. Furthermore, when and where this coordinator functions is not clear. Vg1 plasmid injections can also orient the L-R axis suggesting this process could occur after mid-blastula transition (Hyatt and Yost 1998).
A role for gap junction communication
One process that may function early in the establishment of the L-R axis upstream of the role of the node has been shown to be conserved in both frog and chick. In Xenopus gap junction communication (GJC) shows an early asymmetry with GJC being more active on the dorsal versus the vegetal halves of the embryo (Levin and Mercola 1998b). Inhibition of GJC in Xenopus by pharmacological agents or misexpression of connexin constructs leads to an increase in heart looping defects (Levin and Mercola 1998b). Likewise antibodies or antisense oligonucleotide treatment against connexin subunit Cx43 in chick can inhibit GJC communication, cause bilateral expression of shh and nodal, and thus effect normal heart situs (Levin and Mercola 1999). These treatments also prevent nodes that regenerate after ablation from establishing proper asymmetric gene expression of shh or nodal, suggesting GJC functions in transferring L-R information to the node (Levin and Mercola 1999). Mutations in the connexin subunit Cx43 have been found in some human patients displaying heterotaxia (Britz-Cunningham et al. 1995, but also see Gebbia et al. 1996), and expression of the mutant human Cx43 in Xenopus can induce heterotaxia, suggesting GJC may be important in establishing the human L-R axis as well (Levin and Mercola 1999). Cx43 mutant mice have not been reported to have L-R defects, but this could be due to the ability of other connexins to functionally substitute for the loss of Cx43 (Lo 1999). It is not clear how GJC functions in L-R axis establishment It is conceivable that gap junctions allow unidirectional transport of small molecules, resulting in the accumulation of a L-R determinant on one side. For GJC to be asymmetric in the embryo, there must be a barrier, or "zone of isolation" to prevent GJC from traveling back from one side to the other. In Xenopus this is achieved by preventing GJC communicationfrom occurring in the ventral blastomeres (Levin and Mercola 1998b). In chick GJC there is a lack of gap junctionsin the midline, thus acting as the zone of isolation(Levin and Mercola 1999). If the model proposed above iscorrect, it implies that the initial break in symmetrymust occur upstream of GJC to result in asymmetrictransport of the L-R determinant. To clearly establish the role of GJC in L-R patterning, it will be crucial to isolate the potential L-R determinant transported via gap junctions.
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