Winning RS et al. (2002), EphA4 catalytic activity causes inhibition of R...

XB-ART-7273

Differentiation 2002 Mar 01;701:46-55. doi: 10.1046/j.1432-0436.2002.700105.x.

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EphA4 catalytic activity causes inhibition of RhoA GTPase in Xenopus laevis embryos.

Winning RS , Ward EK , Scales JB , Walker GK .

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The Eph family of receptor tyrosine kinases is involved in limiting cell and tissue interactions via a repulsive mechanism. The mechanism of repulsion involves reorganizing the actin cytoskeleton, but little is known of the molecular components that connect the receptor to the actin cytoskeleton. Recent studies in retinal ganglion cells have demonstrated that EphA4 activates the small GTPase Rho. We have investigated the involvement of Rho in signaling downstream from EphA4. As a model system, we have used a chimeric receptor called EPP that we express and activate in early Xenopus embryos. Previous studies demonstrated that EPP activation leads to loss of cell-cell adhesion and change in cell shape, plus loss of aspects of cell polarity in epithelial cells, such as apical microvilli and the apical/basolateral boundary. In this study, we show that injecting inhibitors of Rho GTPases into early Xenopus embryos produces a phenotype very similar to that resulting from EPP activation. More importantly, expression of a constitutively active form of Xenopus RhoA (XRhoA) concurrent with activated EPP rescued embryos from the loss of cell-cell adhesion and change in cell shape associated with EPP. These data argue that, in contrast to the case in retinal ganglion cells, EphA4 in early Xenopus embryos acts to inhibit RhoA, suggesting that this receptor may regulate Rho differently (and therefore affect the cytoskeleton differently) in neuronal and non-neuronal cells. Furthermore, overexpression of ephexin, a novel guanine nucleotide exchange factor for Rho family GTPases, also blocks EPP-induced dissociation. This suggests that EphA4, which has been demonstrated to activate ephexin in cultured neuronal cells, may also target Rho GTPase via an ephexin-independent pathway.

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Species referenced: Xenopus laevis
Genes referenced: actl6a epha4 rho rho.2 rhoa rhoa.2

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	Fig. 1 Inhibitors of Rho family GTPases cause a loss of cell ad- hesion in early Xenopus embryos. Embryos were injected at one- cell stage with water (A), or with 500 pg of C3 transferase (B), or Toxin A (C). At blastula stage, embryos were fixed in buffered formalin, embedded in plastic, and sectioned. The scale bar in C represents 100 mm.
	Fig. 2 Rho family inhibitors cause change in cell shape and loss of polarity in embryonic cells. Embryos were injected as outlined in Fig. 1. At blastula stage, embryos were fixed in glutaraldehyde, hemisected, and prepared for scanning electron microscopy. A and D, water-injected embryos; B and E, embryos injected with C3 transferase; C and F, embryos injected with Toxin A. Panels A–C show a view of internal cells in the animal hemisphere of the blastula; panels D–F show apical surface of superficial blastula cells. The scale bar in A represents 100 mm and applies to panels A–C; the scale bar in D represents 10 mm and applies to panels D–F.
	Fig. 3 Constitutively active RhoA rescues embryos from the EPP phenotype. Embryos were injected at one-cell stage with the indi- cated RNAs, and prepared as outlined in Fig. 2. A representative RNA; embryo is shown in each panel. A, water-injected embryo; B, em bryo injected with EPP RNA; C, embryo injected with RNAs en coding EPP and TGF-a; D, embryo injected with 500 pg each of RNAs encoding EPP and TGF-a and 250 pg of V14RhoA RNA; E, embryo injected with EPP and TGF-a and 500 pg of V14 RhoA F, embryo injected with EPP and TGF-a and 500 pg of - RNA encoding wild-type RhoA. The scale bar in F represents 100-mm.