DISCUSSION:

The embryos injected with DN-XRhoA mRNA showed inhibition of vegetal rotation that always correlated with impaired formation of Brachet’s cleft on the dorsal side of the embryo. This suggests a role of XRhoA in tissue separation at the Brachet’s cleft that more likely involves mechanical force than differential gene expression. This idea is also supported by earlier studies that found no Brachet’s cleft in the BCR-less embryos. These facts lead to the conclusion XRhoA induces motility in the vegetal cell mass causing the cells to upwell in a rhythm from dorsal to ventral side. As the cells upwell, they shear the mesoderm of the BCF along with them and appose it against the BCR. The ECM of the BCR forms a physical barrier between the mesoderms of the BCR and BCF this gives rise to the Brachet’s cleft as the vegetal cells move further upward, internalizing the mesoderm as they tug at it. Thus vegetal rotation plays an important role in the involution (internalization) of the mesoderm. It is due to the lack of force generated by vegetal rotation that tissue separation at the leading edges does not occur when vegetal rotation is inhibited. Moreover, lack of archenteron formation in the DN-XRhoA embryos also suggests that vegetal rotation might be the primary force involved, at least in the events of early gastrulation.

In addition, the occurrence of large cells and nuclei around the injection site indicate the role of XRhoA in cytokinesis. Since the formation of the contractile ring during cell division also involves myosin filaments that are regulated by ROK (5, 6) this observation leads to the conclusion that XRhoA might be playing its role in vegetal rotation through ROK. This conclusion is also supported by the results of ROK inhibition experiments that also prevent vegetal rotation in a similar fashion to DN-XRhoA.

Furthermore, the occurrence of thick roofs in the vegetal-rotation-inhibited embryos suggests a role for XRhoA in the radial intercalation in the blastocoel roof during early gastrulation that causes the thinning of the BCR (1).

XRhoA is already known to be involved in the process of convergent extension during gastrulation that serves to lengthen the embryo along the anterioposterior axis. This process involves cytoskeletal changes that lead to the mediolateral intercalation of the mesodermal cells, leading the arrangement of mesodermal tissue in a longer thinner array that contributes to an increase in length. This pathway is known as the planar polarity pathway or PCP pathway.

This pathway involves Wnt-11 signal received by frizzled-7 receptor, which then activates a scaffolding protein Dishevelled (Dsh). Dsh in turn activates XRhoA with the help of another protein Daam1. XRhoA activates JNK1 and ROK downstream of it. These two kinases in turn lead to cytoskeletal changes that cause convergent extension movements. In an earlier study (7), the inhibitory effect of DN-XRhoA on convergent extension has been shown to be rescued by wild-type ROK.

Alternatively, another candidate pathway for XRhoA activation in Xenopus is the LPA-GPCR signalling pathway. LPA (lysophosphatidic acid) is a modified phospholipid that acts as a ligand, binding to a G-protein-coupled receptor (GPCR). The GPCR in turn activates an associated G-protein. The α-subunit of that G-protein activates a Rho-GEF, which in turn leads to the activation of Rho itself (5, 6, 8).

An example of this generic pathway in Xenopus is the LPA signalling through XLPA₂ (Xenopus LPA) receptor, which is a GPCR. It was discovered in a recent study that overexpression of XLPA₂ improved wound healing. However, in embryos coinjected with DN-XRhoA mRNA along with XLPA₂ mRNA, there was no improvement in wound healing (5). This lead to the conclusion that XRhoA might be downstream of XLPA₂ in that context.

In future investigations, both the candidate pathways can be tested for their involvement upstream of XRhoA. To test the PCP pathway, the embryos could be injected with a dishevelled mRNA that was dominant negative to the PCP pathway and if it blocked vegetal rotation, it would suggest a possible involvement of the PCP pathway in vegetal rotation. Alternatively if a DN- XLPA₂ mRNA could block vegetal rotation, it would suggest an involvement of the LPA-GPCR signalling pathway in the vegetal rotation.

In both the above instances of dominant negative treatments, we can attempt to rescue the phenotype (if there is one) by coinjecting active Rho or ROK mRNA.

To nail down the factors involved downstream of ROK, we could treat the embryos with myosin inhibitor and if it blocked vegetal rotation, it would confirm the involvement of myosin downstream of ROK in vegetal rotation. However, if this treatment does not inhibit vegetal rotation, then we may consider some other target of ROK to be acting downstream of it in vegetal rotation.