Otte AP et al. (1992), Expression and potential functions of G-protein...

XB-ART-23400

Development 1992 Sep 01;1161:141-6.

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Expression and potential functions of G-protein alpha subunits in embryos of Xenopus laevis.

Otte AP , McGrew LL , Olate J , Nathanson NM , Moon RT .

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During early embryonic development, many inductive interactions between tissues depend on signal transduction processes. We began to test the possibility that G-proteins participate in the signal transduction pathways that mediate neural induction. The expression during Xenopus development of three G alpha subunits, G alpha 0, G alpha i-1 and G alpha s-1, was characterized. The three maternally expressed genes showed different expression patterns during early development. Whole-mount in situ hybridization revealed that all three genes were expressed almost exclusively in the gastrula ectoderm and predominantly in the neuroectoderm in the neurula embryo. In order to investigate the involvement of these proteins in neural induction, we overexpressed the G-protein alpha subunits by injecting the G alpha mRNAs into fertilized eggs. Overexpression of G alpha s-1 increased the ability of gastrula ectoderm to become induced to neural tissue approximately four-fold. Overexpression of G alpha 0 and G alpha i-1 had less pronounced effects on neural competence, and inhibition of the G alpha 0 and G alpha i-1 proteins by pertussis toxin did not change the neural competence of the exposed gastrula ectoderm. Overexpression of the G alpha 0 and G alpha i-1 genes did, however, inhibit the normal disappearance of the blastocoel during gastrulation, suggesting a role for these G-proteins in regulating this process. The data also suggest a specific role for the G alpha s subunit in mediating the initial phases of neural induction.

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Species referenced: Xenopus laevis
Genes referenced: b4galt1.1 b4galt1.2 gnai2 gnao1 gnas ncam1 prph tbx2 trg ugcg

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	Fig. L. Expression of Gtxo, Gcri-r and Gcrt transcripts through early Xenopus development. (A) Antisense probes for the Xenopus Goo, Gcri-r and Gcrs genes (Olate et al., 1989) were used for northern analysis of total RNA (20 trg) isolated from the indicated developmental stages (Nieuwkoop and Faber, 1967). The filter was reprobed with a probe for 55 ribosomal RNA.
	Fig. 2. Localization of Gao, Gai-1 and Gas transcripts in early gastrula and neurula embryos. Whole-mount in situ hybridization was performed on stage-10 early gastrula (A, B, C) and stage-1 4 early neurula (D, E, F) albino embryos. In gastrula embryos, the dorsal (D) and the ventral side (V) side of the embryo are indicated. The anterior (ANT) to posterior (POST) orientation of the neurula embryos, the position of the archenteron (ARCH) and the neural pl ate (N) are indicated. The stained embryos were subsequently sectioned (G, H, I, earl y gastrula and J through 0 , early neurula). The blastocoel (BL) of gastrula embryos (G, H, I), the neuroectoderm (N), the underlying dorsal mesoderm (M) (J, K, L) and the ventral ectoderm (VE) (M, N, 0 ) are indicated. Bar shown in G, 100um for each panel.
	Fig. 3. Effects of injected Gcxo, Gai-r and Gcrs mRNA on the neural competence of early gastrula dorsal and ventral ectoderm. (A) Fertilized eggs were injected with 50 pg Gcxo, Gcri-r and Gcr' mRNA and cultured to stage-10 early gastrula. Recombinates were made between dorsal mesoderrn and stage- 10 dorsal or ventral ectoderm (Otte and Moon, 1992). Recombinates were cultured to stage 22 before total RNA was isolated. The expression of neural specific genes in l0 pg RNA was monitored by RNAase protection, using the probes for the neural specific genes XIF3 and N-CAM. The level of 55 transcripts was monitored by RNA blot analysis of these samples. (B) Control embryos (lanes I and 3) and embryos that were injected with 50 pg Go" or Gcri-r mRNA (lanes 2 and 4) were cultured to stage l0 before homogenization. ADP-ribosylation was carried out in the presence of pertussis toxin and [32p]NAD+. After labeling, the Goo or Gcri- r proteins were immunoprecipitated using antibodies against Goo protein (lanes I and 2) or Gcri- r protein (lanes 3 and 4) and separated on an SDS -lU%o polyacrylamide gel.
	Fig. 4. Effects of overexpressing Gtb, Gai-r and Gq6 rnRl.IA on the development of the blastocoel. (A) Fenilized eggs werc injected wittt 100 pg Goo, Gai-r or Gcs mRNA and cultured to neurula stage. Embryos injected with Go€ rnRNA developed normally with the neural plate facing upwards. The embryos injected with Goo or Gai-r rnRl.[A hrned upside down, resulting in the ventral side facing upwards. (B) The residual blastocoel of an embryo that was injected with Gai-r rnRNA was punctured and the embryo was turned, to show that the neural plate (N) developed normally. (C) The blastocoel of embryos that were injected with G% or Gcri-r mRNA remained as a blister on the ventral side of neurula embryos up to stage 20. (D) A normal stage-l5 embryo was fixed and cut in half to show the development of the archenteron (ARCH) and disappearance of the blastocoel (BL). (E) A stage-l5 embryo that had been injected with Gai-r mRNA was fixed and cut in half to show the retention of the blastocoel (BL). Note the absence of a normally developed archenteron. The endoderm is pushed against the dorsal mesoderm, underlying the neural plate.