4.3.1.1 Injected zebrafish embryos are indistinguishable from wild-type sibs
I show that the overexpression of either wild-type human RhoA or, perhaps more surprisingly, its dominant negative counterpart has no effect on zebrafish embryogenesis. The embryos pass through epiboly and gastrulation, processes both thought to be heavily reliant on the correct and precise movement of the cells and remodelling of their underlying actin cytoskeleton, as normal. There are no effects on the later processes of segmentation, both in the somites of the trunk and the
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régionalisation of the hindbrain, as seen by the regular, undisturbed morphology of both; nor are there any later effects on the patterning and outgrowth of the central nervous system - staining with the anti-tubulin antibody shows a normal axon scaffold with all projections reaching their correct targets. The injected embryos are, in all aspects considered, phenotypically wild-type. In support of these data, embryos treated with the Rho-kinase blocking agent, Y-27632, complete epiboly unperturbed (K. Woolley, unpublished data), implying at some level a redundancy of function for Rho in the early embryo.
4.3.1.2 Overexpression o f these constructs in Drosophila blocks cellularisation and dorsal closure
These results conflict with data gleaned from similar experiments in other organisms. The majority of the work pursued in this area has been carried out in
Drosophila, where differing effects are seen when Rho function is interfered with at differing stages of development. A dominant negative DRhol (the homologue of RhoA) construct expressed widely in the early embryo under the control of the nanos promoter produces obvious defects in gastrulation - there is a delay in the formation of the ventral furrow which subsequently fails to extend at its posterior end. The midgut fails to invaginate and germband extension also fails (Barrett et al.,
1997). This lethal phenotype is remarkably similar to the phenotype generated by knocking out the function of the upstream activator, DRhoGEFl, or those phenotypes seen in the folded gastrulation {fog) and concertina {eta) mutants, fog is a secreted extracellular ligand, whereas eta is the G a subunit of a heterotrimeric protein linked to the fog receptor. Hence, DRhoGEFl is postulated to act as the signalling link between receptors at the cell surface and the Rho cascade. Mutations at any point in this pathway cause defects in the cell shape changes required for gastrulation. Similar results are found in the examination of the role of RhoA in dorsal closure in Drosophila, where expression of an N19RhoA transgene has dramatic effects on the cytoskeleton of the leading edge cells (Harden et al., 1999), giving rise to a segmentally repeated loss of myosin and a loss of contraction of these cells. Embryos homozygous for a lethal P-element insertion abrogating Rhol function display a phenotype in which dorsal closure does occur, but in a disorganised fashion - cells at the midline are inappropriately shaped, some being stretched, others pinched together. There is also a failure in head involution in these embryos, showing that Rhol functioning is essential for Drosophila embryonic development (Magie et al., 1999).
Microinjection of protein reagents corresponding to the dominant negative forms of human RhoA or the C3 exoenzyme into the posterior poles of cellularizing Drosophila embryos gives a spatiotemporally specific analysis of Rho function, which is more finely-tuned than the widespread expression mentioned previously (Crawford et al., 1998). Within minutes of injection of either protein, there is a halt in the invagination of nascent cleavage furrows brought about by disruption of the cytoskeleton, with a breakdown of the regular, hexagonal actin array into aggregates. There is a progressive disorganisation of the nuclei, which fall away from the surface of the embryo as a presumed secondary consequence of the cortical actin breakdown. These effects are dose-dependent with respect to the amount of protein injected and can be ameliorated by pre-injection of the embryo with the wild-type or constitutively active versions of RhoA, neither of which generate a phenotype when injected alone - injected embryos pass through cellularisation and gastrulation as usual.
4.3.1.3 Modifying expression o f RhoA leads to epithelial polarity defects
Restricted overexpression of a wild-type transgene in the Drosophila eye imaginai disc in the cells posterior to the morphogenetic furrow gives rise to a particular eye phenotype (Hariharan et al., 1995). In the presence of one copy of this transgene, the eye appears normal but will deform easily under mild pressure, and has an irregular shape and arrangement of rhabdomeres. When a double dose of the transgene is received, the eye becomes very rough and there is a considerable disruption of the ommatidial architecture - the ommatidial cytoskeleton is disorganised and the later differentiation of the photoreceptor and pigment cells fails to occur correctly. These results were taken to mean that an increase in Rho expression in cells, although not affecting the determination of cell fate, impedes the ability of the actin cytoskeleton to tolerate shape changes and causes cells to become abnormal in their morphology.
Elsewhere, it is reported that strong loss of function RhoA alleles present a tissue polarity phenotype similar to that of the frizzled (Fz) and dishevelled (dsh) mutants which are downstream components of the Wingless signalling pathway (Fanto et al., 2000), A gain of function in these genes can be dominantly suppressed by a reduction in the amount of functioning RhoA. RhoA is also required for the polarity and number of hairs present on the wing i.e. RhoA participates in signalling from membrane-bound receptors to the nucleus in order to generate polarity in epidermal structures (Strutt et al., 1997). This dynamic developmental system which
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requires cells to move and rotate in a coordinated way is achieved by modification of the cytoskeleton by small GTPases accompanied by Fz-induced nuclear signalling.
4.3.1.4 There is no clear reason for the lack o f phenotype seen in zebrafish
By reviewing the data provided from such experiments in Drosophila, it is clear that no homologous phenotypes are generated by over-expression of wild-type or dominant negative RhoA in the zebrafish embryo. Embryos do not lag behind or fail to complete gastrulation; nor do they exhibit any problems with epithelial patterning or muscle induction (see 1.1.6.1). These results are not concordant with those from other organisms and speculation might be made as to why this is so. There may be an inefficient processing of the injected RNA, albeit an unlikely suggestion in the light of the effects seen with the other injected constructs and the widespread expression of the Myc tag. It may transpire that there is such a high level of interacting factors present in the embryo that the supposed sequestering of exchange factors and blocking of endogenous function does not occur at the level of exogenous protein it is possible to generate through the technique of mRNA injection - a way to test this theory would be to inject the protein reagents directly into fertilised eggs. The lack of an obvious phenotype is unlikely to stem from the use of the human homologues of the genes, as these have been found to function in Drosophila and Xenopus. It may well be a combination of the specificity of the dominant negative RhoA and the redundancy of function of shared homologues in the zebrafish that brings about this apparent anomaly.
4.3.2 Expression o f constitutively active RhoA causes fusion o f the eyes and