Wu C et al. (2000), RGS proteins inhibit Xwnt-8 signaling in Xenopu...

XB-ART-10862

Development 2000 Jul 01;12713:2773-84. doi: 10.1242/dev.127.13.2773.

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RGS proteins inhibit Xwnt-8 signaling in Xenopus embryonic development.

Wu C , Zeng Q , Blumer KJ , Muslin AJ .

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RGS family members are GTPase activating proteins (GAPs) that antagonize signaling by heterotrimeric G proteins. Injection of Xenopus embryos with RNA encoding rat RGS4 (rRGS4), a GAP for G(i) and G(q), resulted in shortened trunks and decreased skeletal muscle. This phenotype is nearly identical to the effect of injection of either frzb or dominant negative Xwnt-8. Injection of human RGS2, which selectively deactivates G(q), had similar effects. rRGS4 inhibited the ability of early Xwnt-8 but not Xdsh misexpression to cause axis duplication. This effect is distinct from axin family members that contain RGS-like domains but act downstream of Xdsh. We identified two Xenopus RGS4 homologs, one of which, Xrgs4a, was expressed as a Spemann organizer component. Injection of Xenopus embryos with Xrgs4a also resulted in shortened trunks and decreased skeletal muscle. These results suggest that RGS proteins modulate Xwnt-8 signaling by attenuating the function of a G protein.

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Species referenced: Xenopus
Genes referenced: dvl1 frzb gnaq myod1 odc1 rgs2 rgs4 tbxt wnt8a

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Fig. 2. RGS4 RNA injection at the 2- cell stage perturbs skeletal muscle and retinal development. (A,B) Immunocytochemical staining of skeletal muscle in a stage-28 embryo injected with control fs- RGS4 RNA (A) or with wild-type RGS4 RNA (B). The section was stained with the muscle-specific antibody 12/101 (Kintner and Brockes, 1984), and bound antibody was visualized with a red color substrate. The section was then stained with Hematoxylin. Arrows indicate skeletal muscle. (C,D) Histological section through the eye of a stage-28 embryo injected with control fs-RGS4 RNA (C) or wild-type RGS4 RNA (D) and stained with Hematoxylin. Note the infoldings of the pigmented (black) presumed retinal cells in the wildtype RGS4 RNA-injected embryo. (E,F) Expression of XmyoD in gastrula-stage embryo injected with control fs-RGS4 RNA (E) or wildtype RGS4 RNA (F) and visualized by whole-mount in situ hybridization. (G,H) Expression of Xbra in gastrula-stage embryo injected with control fs-RGS4 RNA (G) or wild-type RGS4 RNA (H) and visualized by whole-mount in situ hybridization.

Fig. 9. Developmental expression pattern of Xrgs4a. (A-H) Spatial expression pattern of Xrgs4a. Whole-mount in situ hybridization analysis of gene expression in albino embryos at the gastrula, neurula and tailbud stages. (A,C,E,G) In situ hybridizations were performed with an antisense Xrgs4a-probe. (B,D,F,H) In situ hybridizations were performed with a sense Xrgs4a-probe. (A,B) Vegetal view of stage-10.25 embryos. (C-H) Lateral view of stage- 16 (C,D), stage-25 (E,F) and stage-35 (G,H) embryos. Anterior is right. Xrgs4a is expressed in the dorsal lip of the blastopore, with expression extending over the dorsal aspect of the gastula-stage embryo (A). Xrgs4a is expressed in the neural folds of neurula-stage embryos (C,E). Xrgs4a is expressed in the head, retina, spinal cord, cement gland and heart of the tailbud-stage embryo (G). (I) Timing of Xrgs4a gene expression. RNA was purified from whole embryos at the indicated embryonic stages (Nieuwkoop and Faber, 1967). RTPCR was performed by use of specific primers that recognized Xrgs4a or ornithine decarboxylase (ODC), as described in Materials and Methods. The 40â RT lane contained all reagents except reverse transcriptase, and was used as a negative control; ODC was used as a loading control.