Kim GH and Han JK (2005), JNK and ROKalpha function in the noncanonical W...

XB-ART-2250

Dev Dyn 2005 Apr 01;2324:958-68. doi: 10.1002/dvdy.20262.

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JNK and ROKalpha function in the noncanonical Wnt/RhoA signaling pathway to regulate Xenopus convergent extension movements.

Kim GH , Han JK .

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The Wnt/planar cell polarity (PCP) pathway plays a critical role in wing, eye, and sensory bristle development of Drosophila and in convergent extension (CE) movements during vertebrate gastrulation. In Drosophila, Jun N-terminal kinase (JNK) and Rho-associated kinase (ROK) participate in RhoA-mediated PCP pathway during eye and wing development. In mammalian cells, Rac1 and Cdc42 but not RhoA are required for JNK activation by Wnt/PCP signals. However, there has been no evidence that Rho GTPases regulate JNK activation in Wnt/PCP pathway during Xenopus CE movements. Here, we report that Xenopus RhoA (XRhoA), but not Xenopus Cdc42 (XCdc42), is essential for JNK activation downstream of the Wnt/PCP pathway during Xenopus CE movements, and the phenotypic effect of loss of XRhoA function was rescued by Xenopus JNK1 (XeJNK1). In addition, XRhoA rescues the inhibition of CE movements by the DEP domain deletion mutant of Xenopus Dsh (Xdsh-DeltaDEP), which has dominant negative (DN) effects on JNK activation, and the PDZ domain deletion mutant of Xdsh (Xdsh-DeltaPDZ). Moreover, we demonstrate that Xenopus Rho-associated kinase alpha (xROKalpha), which is expressed mainly in mesoderm and ectoderm that undergo extensive cell rearrangements, regulates CE movements without affecting gene expression, and injection of xROKalpha rescued the inhibition of CE movements caused by DN XRhoA. Finally, we show that ROKalpha and JNK synergistically rescued embryos overexpressing DN XRhoA, which exhibit gastrulation defects, although ROKalpha is not required for JNK activation. Together, these data suggest that JNK and ROKalpha function in the noncanonical Wnt/RhoA pathway to regulate Xenopus CE movements.

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Species referenced: Xenopus
Genes referenced: a2m cdc42 chrd col2a1 daam1 dvl1 dvl2 gsc jun mapk8 myod1 ncam1 not odc1 otx2 rac1 rho rho.2 rhoa rhoa.2 rock2 tbxt

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Figure 4. Xenopus Rho-associated kinase alpha (xROK alpha ) regulates convergent extension (CE) movements without affecting gene expression. A: Temporal expression pattern of xROK alpha . Developmental stages are shown above each lane. Ornithine decarboxylase (ODC) was used as a loading control. B-D: Spatial expression pattern of xROK alpha . B: Expression pattern of xROK alpha at a stage 10.25 gastrula (dorsovegetal view). An arrow indicates the dorsal lip of blastopore. C: Sagittal section of a stage 10.25 gastrula. xROK alpha transcripts were mainly expressed in the dorsal mesoderm and ectoderm above the dorsal lip. An arrow indicates the dorsal lip of blastopore. D: At the stage 35 tadpole, xROK alpha was expressed in head and tail ectoderm and mesoderm. E: Dorsal two blastomeres of four-cell stage embryos were injected with xROK alpha mRNA (500 pg) and allowed to develop to stage 33 (lateral view). Two blastomeres of four-cell stage embryos were injected dorsally with 750 pg of xROK alpha . Keller explants were dissected at stage 10.5 and cultured until siblings were reached at stage 19. WT, wild-type. F: Quantitation of relative rescue in whole embryos for E. G: Quantitation of relative elongation of Keller explants compared with controls for E. In the Student's t-test, all differences are statistically significant (P < 0.05). These examinations were performed more than three times. Others indicate truncated axis and mild kinked axis. H,I: Analysis of reverse transcriptase-polymerase chain reaction demonstrated that xROK alpha does not affect dorsal cell fate specification in Keller explants cultured to stage 12 (H) and dorsoventral patterning of the mesoderm in Keller explants cultured to stage 22 (I). W.E indicates whole embryo; -RT indicates no reverse transcriptase control. ODC is the loading control.