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Three theoretical models for the determination o f LR asymmetry in vertebrae have been postulated. The first two, those o f Brown and Wolpert and Almirantis are broadly similar. The third is completely novel and draws from work in yeasts.

Browns and Wolperts Model

This model has three components which are termed conversion, random generation o f asymmetry and interpretation. Conversion refers to the conversion o f molecular handedness into handedness at a cellular and multicellular level. Handed positional information is derived from an handed or chiral molecule (called the "F" molecule) which must be tethered in respect to the AP and DV axes. This molecule influences the transport other cellular constituents and creates a difference in polarity between cells to the right and cells to the left o f the midline (Figure 3). This difference is stabilised on the right side o f the embryo while the left remains capable o f reeducation. The stable property consistently biases the mechanism for the random generation o f asymmetry, which could be a morphogen gradient, to one side. Individuals tissues interpret the difference between the two sides by developing different structures on left and right. Genetic control o f the left right axis can break down at various points in the pathway with different predicted phenotypic results. Absence o f the handed molecule or its receptor will lead to lack o f a consistent bias

and therefore random generation o f asymmetry as in Kartageners syndrome or the iv/iv mouse. . The phenotype o f the iv mouse can be explained according to Brown and Wolpert's model as a failure o f conversion. Thus there is no consistent bias to the mechanism for the random generation o f asymmetry and situs is randomised. The mutation may lead either to absence o f the F molecule or its failure to align to the AP

and DV axes. Consistent situs inversus could be produced by tethering the

asymmetric molecule in the wrong direction so reversing the bias. If a specific concentration range o f morphogen is important for specifying right or left sided structures, by changing either the concentration o f morphogen or morphogen receptor, a left sided pattern might be induced on the right resulting in reversal of normal asymmetry or in bilateral symmetry. In Brown and Wolperts terms this is an example o f consistently erroneous interpretation. The threshold o f response of difference tissues to the concentration o f morphogen may differ both temporally and spatially and so subtle changes in morphogen concentration may affect one organ but not another leading to discordance of organ laterality or heterotaxia. . The consistently inverted left-right polarity might result from a loss o f function mutation which unmasks a default pathway as in the recessive snail mutant, sinistral. Alternatively the F molecule might become tethered in the opposite direction thus reversing the conversion process.

Almirantis' Model

Almirantis proposes a very similar model to Brown and Wolpert. Whereas they consider that the combination o f F molecule alignment with the polarisation o f cells is enough to confer a difference between left and right sides o f the midline Almirantis postulates that this interaction triggers the creation o f a specific left-right orientated morphogenic gradient which once created is independent o f the molecular micro structure. This LR gradient is likely to be exponential.

Klar's Model of non random chromosome segregation

Klar's model is based on the programmed pattern o f cell type change in fission yeast which results from inheritance o f specific chromatids o f the parental chromosome. The model proposes that DNA replication produces different chromatids and that specific chromatids o f both homologues are nonrandomly segregated to daughter cells to specify the left right axis o f the embryo. This model predicts that left - right information is cell-autonomous in mammalian embryos. However this is not the case. Labelling single cells in any region of mouse ectoderm shows that even at late primitive streak stages daughter cells are found on both sides o f the midline (Lawson et al. 1991).

Anterior

Posterior

1.3.2.2 Amphibia

Pre Molecular Experiments

Since the early 1900s classical experiments involving various nonspecific nonsurgical treatments as well as microsurgical manipulations (Wehrmaker, 1969) have been shown to induce cardiac and visceral situs inversus in amphibian embryos. M ost o f the early work was done in the urodele Triturus which has an unusually high frequency o f spontaneous situs inversus (1-2%) compared to other amphibians like Xenopus and indeed compared to man (1 in 10,000). In Triturus any treatment which produces abnormalities in sidedness always leads to a spectrum o f reversal ranging from almost normal situs to complete situs inversus (Wehrmaker, 1969). Perhaps the best known experiment was that o f Spemann and Falkenberg in 1919 who found that on median constriction o f the egg during cleavage or blastula stage the twin that arises from the right half is often reversed. However this was never observed in more than 50% o f the right sided twins. This led Wilhelmi to suggest that the orientation o f situs was developing randomly in the right twin (Brown and Wolpert, 1990).

These classical experiments have been extended and refined using the anuran Xenopus laevis as a model system. The first obvious breaking o f midline symmetry in Xenopus is heart looping and investigations have tended to focus on the effects o f various experimental manipulations on this process.

In Xenopus, cells destined to form the heart arise from a pair o f dorsolateral mesodermal primordia through inductive interactions with the Spemann Organizer region (Yost, 1995a). At the end o f gastrulation the cardiac primordia move from a dorsolateral position to the ventral midline across an extracellular matrix on the basal surface o f the embryonic ectoderm. At the ventral midline the two primordia fuse to form a single sheet o f cells which later forms the cardiac tube. Bilateral symmetry is broken at the late neurula - early tailbud stage when the cardiac tube twists and loops to the right forming an S shaped tube. Failure to specify the left and right cardiac

primordia should prevent heart looping whereas reversing LR polarity will result in a left sided heart loop, that is, cardiac situs inversus.

Four periods o f embryonic development during which experimental perturbation results in altered left-right development o f the heart have been identified (Yost, 1995a). The first is during the first cell cycle concurrent with DV axis determination. The amphibian egg is cylindrical with the animal and vegetal poles defined by the relative postions o f the nucleus and the mitochondrial mass in the oocyte (Gurdon, 1992) . The plane o f the DV axis is determined by the sperm entry point. This induces a rotation o f the subcortical cytoplasm driven by arrays o f microtubules near the surface o f the vegetal hemisphere. The dorsal midline is established at the meridian along which the subcortical cytoplasm moves to the greatest extent vegetally (Yost, 1991). UV irradiation o f the vegetal pole o f the egg destroys the microtubule array and prevents cytoplasmic rotation producing embryos which fail to develop any dorsoanterior structures. UV treated eggs can be rescued inducing cortical rotation by manual tilting. Around 25% o f rescued eggs develop into embryos with complete reversal o f the LR axis (Yost, 1991). This is likely to be secondary to impaired dorsoanterior development. In birds and mammals the AP and DV axes are not apparent until blastocyst stages. In the mouse the polarity o f the AP axis is not specified until after implantation (Gardner et al. 1992) and cells are not committed to left and right sides even at early primitive streak stages (Lawson et al. 1991). The majority o f human monozygotic twins split at the early cleavage stages develop normally.

The second critical time period is late blastula to early gastrula stage. Disruption o f the extracellular matrix (ECM), which is deposited on the basal surface o f the animal pole ectoderm at this stage perturbs LR axis patterning (Yost, 1992). If the entire ECM is eliminated each organ primordium breaks symmetry in an orientation that is random and independent o f the orientations o f the other primordia. There are two possible ways in which the ECM might impart LR information to the primordia as

they migrate across it. A secreted factor could be anchored within the matrix in an

asymmetric distribution. (Yost, 1995a). In this case one would predict the

asymmetric expression o f a morphogen or receptor secreted by the ectoderm and held in the ECM at this time. An alternative explanation for the role o f the ECM draws on the hypothesis that a molecule with innate molecular handedness influences LR development (Brown and Wolpert, 1990) . Yost suggests that the ECM fibrils may have molecular handedness which can be detected by the primordial cells as they move across the matrix (Yost, 1995a). For the cells to interprète the matrix differently depending on whether they approach from right or left the fibrils must be aligned with respect to the AP and DV axes.

Inhibition o f proteoglycan synthesis during a narrow time window from late gastrula to early neurula has a different effect. It totally eliminates looping o f the cardiac tube. The critical period coincides with the migration o f the cardiac primordia to the ventral midline (Yost, 1990).

Several experimental approaches suggest that dorsal midline cells, which form the Organiser, and the notochord are essential in regulating cardiac LR asymmetry during the neurula stage o f development (Danos and Yost, 1995). Treatments which curtail dorso-anterior development, for example ectopic expression o f Xwnt-8 in the dorsal- most blastomeres, cause an increase in cardiac LR reversals (Danos and Yost, 1995). These experiments do not identify the specific dorsal midline cells influencing LR asymmetry. Extirpations o f midline cells including the notochord during mid neurula stages result in randomisation o f cardiac looping after Organizer activity has diminished(Danos and Yost, 1995). Explants o f precardiac mesoderm form cardiac tubes which loop in vitro. The orientation o f looping is randomized in explants from early neurula stages but is correctly orientated from explants o f late neurula stages(Yost, 1995b).

The notochord is known to have inductive properties (Placzek, 1995) and may produce a signal influencing LR asymmetry in cardiac cells either directly or indirectly.

Gene Expression Studies

Gene expression studies in Xenopus confirm the conclusions from experimental manipulations. There is evidence for molecular LR asymmetry, involving theTGpp family gene, V g l, as early as the 16 cell blastula stage in Xenopus (Hyatt et al. 1996). V gl is a Xenopus maternal gene which is known to be important in dorsoanterior development. It is regulated by cleavage o f the precursor molecule to produce the mature active Vgl protein. Altered expression o f Vgl on the right side o f 16 cell embryos or disruption o f endogeneous V gl signalling on the left side randomizes cardiac and visceral LR orientation (Hyatt et al. 1996). Cell-lineage directed expression o f V gl protein can also fully invert the left-right axis, and can “rescue” a perturbed left-right axis in the right hand twin o f conjoint twins (Hyatt and Yost, 1998). Hyatt and Yost postulate that Vgl acts as the “left-right coordinator” . In normal development activation o f the LR coordinator initiates the left identity o f the left side and the identity o f the right side by the production o f antagonistic signals from the left.

What biases processing o f V gl? A group o f dorsovegetal cells known as the Nieuwkoop centre emit cell signalling molecules which lead to mesoderm induction and dorsoanterior axis formation. The Nieuwkoop centre can be mimicked by mRNAs o f the Wnt signalling pathway. If these are injected into the L or R ventral vegetal blastomere at the 8 -16 cell stage a 2nd organising centre and complete secondary axis is induced. Induction o f a 2nd organising centre in this way seems to be enough to generate normal LR asymmetry with respect to DV and AP axes. In other words there is no global LR pattern throughout the embryo; the ectopic organiser has innate

LR asymmetry (Nascone and Mercola, 1997). It is likely that molecules produced from the organiser, possibly Wnt pathway molecules, bias V gl processing.

Expression o f Xnr-1, a member o f the TGFp superfamily, becomes strikingly asymmetric during neuralation in Xenopus (Lowe et al. 1996). At first, post gastrulation, it is detected in a bilaterally symmetric pattern adjacent to posterior limit o f the notochord but subsequently it is expressed only in the left lateral plate mesoderm. This asymmetric left sided expression pattern depends both on left sided Vg-1 signalling (Nascone and Mercola, 1997)and on the presence o f dorsal midline structures, including notochord and neural floor plate. These structures normally repress Xnr-1 expression on the right side o f the embryo. Abnormal symmetric Xnr-1 expression patterns correlates well with cardiac reversal rates in both control and experimentally treated Xenopus embryos (Lohr et al. 1997). Similar asymmetric expression patterns o f nodal the mouse and chick homologue are seen in the left lateral plate mesoderm during neuralation in chick and mouse suggesting conservation o f function o f nodal in LR determination.