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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.
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.
