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Dev Biol
2003 May 15;2572:302-14. doi: 10.1016/s0012-1606(03)00067-8.
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Activation of Gbetagamma signaling downstream of Wnt-11/Xfz7 regulates Cdc42 activity during Xenopus gastrulation.
Penzo-Mendèz A
,
Umbhauer M
,
Djiane A
,
Boucaut JC
,
Riou JF
.
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Wnt-11/Xfz7 signaling plays a major role in the regulation of convergent extension movements affecting the dorsal marginal zone (DMZ) of gastrulating Xenopus embryos. In order to provide data concerning the molecular targets of Wnt-11/Xfz7 signals, we have analyzed the regulation of the Rho GTPase Cdc42 by Wnt-11. In animal cap ectoderm, Cdc42 activity increases as a response to Wnt-11 expression. This increase is inhibited by pertussis toxin, or sequestration of free Gbetagamma subunits by exogenous Galphai2 or Galphat. Activation of Cdc42 is also produced by the expression of bovine Gbeta1 and Ggamma2. This process is abolished by a PKC inhibitor, while phorbol esther treatment of ectodermal explants activates Cdc42 in a PKC-dependent way, implicating PKC downstream of Gbetagamma. In activin-treated animal caps and in the embryo, interference with Gbetagamma signaling rescues morphogenetic movements inhibited by Wnt-11 hyperactivation, thus phenocopying the dominant negative version of Cdc42 (N(17)Cdc42). Conversely, expression of Gbeta1gamma2 blocks animal cap elongation. This effect is reversed by N(17)Cdc42. Together, our results strongly argue for a role of Gbetagamma signaling in the regulation of Cdc42 activity downstream of Wnt-11/Xfz7 in mesodermal cells undergoing convergent extension. This idea is further supported by the observation that expression of Galphat in the DMZ causes severe gastrulation defects.
Fig. 7. Expression of exogenous Gβ1 and Gγ2 subunits inhibits convergent extension in a Cdc42-dependent way. Messenger RNA encoding Gβ1 and Gγ2 was microinjected alone or in combination with N17 Cdc42, Gαi2, or Gαt mRNA. Activin-treated animal cap elongation was scored as described in Fig. 5 (A–E). Elongation observed in uninjected controls (A) is significantly inhibited as a result of Gβ1γ2 expression (B). Coexpression of N17Cdc42 (C) clearly restores elongation. Elongation inhibition caused by Gβ1γ2 expression is also rescued by Gαi2 (D) or Gαt (E), showing that it specifically results from the hyperactivation of Gβγ signaling. (F) RT-PCR analysis of mesodermal marker gene expression. Expression of mesodermal markers in response to activin treatment is not modified by Gβ1γ2 expression, indicating that Gβ1γ2 expression directly affects morphogenetic movements and not tissue specification.
Fig. 8. Dorsal expressions of Gαt inhibits gastrulation movements. Messenger RNA encoding Gαt was injected dorsally as described in Fig. 6. Embryos were cultured and scored for anomalies of external morphology. Representative examples are shown at early (A, B), late gastrula stage (C, D), neurula stage (E, F), and tailbud stage (G–I). (A, C, E, G) Sibling controls. (B, D, F, H, I) Gαt-injected embryos. Blastopore closure is strongly affected as a result of Gαt expression. Even when blastopore closure completes, dorsal axis elongation is clearly inhibited (H, I). (J, K) Gαt phenotype specifically results from the inhibition of Gβγ signaling. Blastopore closure and dorsal axis elongation blocked by Gαt expression (J) are restored by coexpression of Gβ1 and Gγ2 (K). (L) Dorsal expression of Gαt does not affect mesodermal or neural patterning. Embryos were fixed at early (st. 10.5) and late gastrula stage (st.13), and were processed for in situ hybridization analysis of mesodermal (Xbra, gsc, Xnot, Otx-2) and neural (Sox-3, Otx-2) marker genes expression. All markers analyzed can be detected in Gαt-injected embryos, showing that Gαt directly interferes with morphogenetic movements, and not with tissue specification. Xnot expression at st. 13 reveals that notochord is present dorsally. However, notochord elongation has been dramatically inhibited, indicating that convergent extension is strongly affected. At st.13, Otx-2 and gsc transcripts are also clearly detected in mesendoderm but at a location which suggests that mesendoderm has failed to undergo normal involution.
