May 1, 2007;
Wnt-5A/Ror2 regulate expression of XPAPC through an alternative noncanonical signaling pathway.
XWnt-5A, a member of the nontransforming Wnt-5A class of Wnt ligands, is required for convergent extension movements in Xenopus embryos. XWnt-5A knockdown phenocopies paraxial protocadherin (XPAPC
) loss of function: involuted mesodermal cells fail to align mediolaterally, which results in aberrant movements and a selective inhibition of constriction. XWnt-5A depletion was rescued by coinjection of XPAPC
RNA, indicating that XWnt-5A acts upstream of XPAPC
. XWnt-5A, but not XWnt-11, stimulates XPAPC
expression independent of the canonical Wnt/beta-catenin pathway. We show that transcriptional regulation of XPAPC
by XWnt-5A requires the receptor tyrosine kinase Ror2
. XWnt-5A/Xror2 signal through PI3 kinase and cdc42
to activate the JNK
signaling cascade with the transcription factors ATF2
. The Wnt-5A/Ror2
pathway represents an alternative, distinct branch of noncanonical Wnt signaling that controls gene expression and is required in the regulation of convergent extension movements in Xenopus gastrulation.
[+] show captions
Figure 1. XWnt-5A Acts Upstream of XPAPC(A) Autoradiograph of 35S-methionine-labeled in vitro translation of a morpholino-sensitive XWnt-5A construct in the absence and presence of XWnt-5A MO shows blocking of XWnt-5A translation by the MO.(B) XWnt-5A knockdown phenocopied the selective inhibition of constriction of XPAPC loss of function in Keller open-face explants.(C) Explants were scored for elongation (upper panel) and constriction (lower panel). XWnt-5A MO was rescued by coinjection of MO-insensitive XWnt-5A RNA and by XPAPC RNA (***p > 0.99 to XWnt-5a MO).(D) In situ hybridization with an XPAPC probe showed downregulation of XPAPC by XWnt-5A MO, but not by XWnt-5A 5MM MO, and was rescued by coinjection of a MO-insensitive XWnt-5A RNA. The injected side is labeled with X-Gal and is indicated with an asterisk.(E) Transcriptional regulation of XPAPC by XWnt-5A and the specificity of the effect were confirmed by real-time RT-PCR.All charts show mean ± SEM (*p > 0.95; **p > 0.99 to controls).
Fig S1 Spatial expression pattern of XWnt5A and XPAPC. XWnt5A and XPAPC RNA localization was analyzed in embryos of stage 10.5, 11 and 12 by whole mount in situ hybridization. XWnt5A was expressed in the deep cells of the ectodermal layer and showed a dorsal bias at early gastrula stages. XPAPC was found specifically in Spemann organizer at stage 10.5 and in the involuting mesoderm at stage 11 and 12. XWnt5A and XPAPC expression overlap in the organizer region at stage 10.5. At stage 11 and 12 XWnt5A is expressed in the deep cell layer adjacent to the XPAPC expressing mesoderm.