Kume S et al. (2000), Galphas family G proteins activate IP(3)-Ca(2+)...

XB-ART-10313

Dev Biol 2000 Oct 01;2261:88-103. doi: 10.1006/dbio.2000.9849.

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Galphas family G proteins activate IP(3)-Ca(2+) signaling via gbetagamma and transduce ventralizing signals in Xenopus.

Kume S , Inoue T , Mikoshiba K .

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During early embryonic development, IP(3)-Ca(2+) signaling transduces ventral signaling at the time of dorsoventral axis formation. To identify molecules functioning upstream in this signal pathway, we examined effects of a panel of inhibitory antibodies against Galphaq/11, Galphas/olf, or Galphai/o/t/z. While all these antibodies showed direct inhibition of their targets, their effects on redirection of the ventral mesoderm to a dorsal fate varied. Anti-Galphas/olf antibody showed strong induction of dorsal fate, anti-Galphai/o/t/z antibody did so weakly, and anti-Galphaq/11 antibody was without effect. Injection of betaARK, a Gbetagamma inhibitor, mimicked the dorsalizing effect of anti-Galphas/olf antibody, whereas injection of adenylyl cyclase inhibitors at a concentration which inhibited Galphas-coupled cAMP increase did not do so. The activation of Galphas-coupled receptor gave rise to Ca(2+) transients. All these results suggest that activation of the Galphas-coupled receptor relays dorsoventral signal to Gbetagamma, which then stimulates PLCbeta and then the IP(3)-Ca(2+) system. This signaling pathway may play a crucial role in transducing ventral signals.

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Species referenced: Xenopus
Genes referenced: actl6a actn1 camp cfb chrd gnaq gnas itpr1 nog tbx2 ventx1.2

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	FIG.1. Western blot analysis of cross reactivities of Ga polyclonal antibodies against Xenopus. Xenopus oocyte crude extracts (20 mg) were loaded onto each lane. Specific affinity-purified polyclonal antibodies against Gaq/11 (Gq/11), Gas/olf (Gs/olf), and Gai/o/t/z (Gi/o/t/z) were used. Single bands (marked by an arrowhead) with strong intensity were detected with antibodies directed against Gaq/11 or Gas/olf proteins, indicating that these antibodies reacted specifically with Xenopus Gaq/11 and Gas/olf. Antibody against Gai/o/t/z also recognized a band with a similar molecular weight.
	FIG. 2. Antibodies against C-termini of Gaq/11, Gai/o/t/z, or Gas/olf all act as inhibitory antibodies in Xenopus embryos. Antibodies were injected at 8 ng per blastomere. Numbers of embryos examined are shown in parentheses. (A) mAChR1-mediated Ca21 mobilization in animal caps was significantly inhibited by injection of antibody directed against the C-terminus of Gaq/11 (aGaq/11), but not by normal rabbit IgG (rIgG). Peak values of intracellular Ca21 concentration ([Ca21]i) in animal caps before (open bars) and after (closed bar, IgG injected; striped bar, aGaq/11 injected) carbachol application are shown as means 6 standard errors of the mean (SEM). P , 0.001, as determined by two-tailed, paired Student’s t test. Numbers of animal caps examined are shown in parentheses. (B) b2AR receptor-mediated adenylyl cyclase activation was significantly inhibited by injection of antibody directed against the C-terminus of Gas/olf (aGas/olf) but not by normal rabbit IgG. Embryos at 2- to 4-cell stage were injected sequentially with FLCRhR at 20 mM, b2AR RNA, and then normal rabbit IgG or anti-Gas/olf antibody. Animal caps of blastula stage (stage 8–9) embryos were excised and subjected to cAMP imaging. Increase in cAMP was observed upon ligand, arterenol application. Time courses of cAMP changes are shown as F520/F580 ratio by calculating the average fluorescence intensity from nearly the entire area of animal caps. Typical time courses of F520/F580 ratio in a control normal rabbit IgG-injected animal cap (top trace) and in an anti-Gas/olf antibody-injected animal cap (bottom trace) are shown. The stippled bars show application of 100 mM arterenol. There was no significant difference between the baseline F520/F580 ratio values of anti-Ga/olf antibody-injected animal caps and those of the control animal caps. P . 0.05, as determined by Mann–Whitney U test. (C) Statistical analysis. Peak amplitudes of changes in [cAMP] (F520/F580 ratio) were compared between anti-Gas/olf antibody-injected animal caps (n512) and control normal rabbit IgG-injected animal caps (n510). Peak changes in [cAMP] are shown as % ratio change normalized by baseline values. Values (% ratio) are expressed as means 6 SEM. Compared to an average of 5% change in peak [cAMP] in control animal caps, animal caps injected with anti-Gas/olf antibody did not show an increase in [cAMP]. P , 0.0001, as determined by Mann–Whitney U test. Numbers of animal caps examined are shown in parentheses. (D) Typical time-course plots of [cAMP] changes in animal caps injected with anti-Gai/o/t/z antibody (aGai/o/t/z; bottom trace) or control normal rabbit IgG (top trace) are shown as F520/F580 ratio. Embryos were injected sequentially with FLCRhR, an RNA cocktail of Gai/o/t/z-coupled mAChR2 and Gas-coupled-bAR RNAs, and then anti-Gai/o/t/z antibody or normal rabbit IgG. Stage 8–9 animal caps were excised and subjected for cAMP imaging. The data were obtained by calculating the average fluorescence intensity from nearly the entire area of animal caps. [cAMP] level is first elevated by bath application of b2AR agonist, arterenol (striped bars). After wash, carbachol (mAChR2 agonist) was applied (filled bars) to measure Gai/o activity (i.e., decrease in [cAMP]). The slope (measured by fitting to a line; gray lines) of the decrease phase of F520/F580 ratio induced by carbachol was larger in animal caps injected with anti-Gai/o/t/z antibody. There was no significant difference in the baseline F520/F580 ratio values and in the rising phase slopes between anti-Gai/o/t/z antibody-injected animal caps and the control rabbit IgG-injected animal caps. P.0.05, as determined by Mann–WhitneyUtest. Gray dotted lines are indicated to clarify the slopes. (E) Statistical analysis. Gai/o-mediated adenylyl cyclase inhibition was significantly blocked by injection of antibody directed against the C-terminus of Gai/o/t/z (n 5 6) but not by normal rabbit IgG (n 5 5). The decrease phase of F520/F580 ratio induced by carbachol was fitted to a line and the slope was calculated and compared. Slope amplitudes (ratio/s) are expressed as means6SEM. P,0.05, as determined by Mann–WhitneyUtest. Numbers of animal caps examined are shown in parentheses.
	FIG. 3. Ventral injection of inhibitory antibodies directed against the C-termini of Gas and Gai/o/t/z, but not that of Gaq/11, showed ectopic axis-inducing activity, with a potency of anti-Gs/olf @ anti-Gai/o/t/z. (A) Histogram of frequency of axis duplication induced by injections of different doses of antibodies into two ventral blastomeres at the four-cell stage. Neither anti-Gaq/11 antibodies (aGaq/11) nor normal rabbit IgG (rIgG) induced an ectopic dorsal axis. Injection of anti-Gas/olf (aGas/olf) or anti-Gai/o/t/z antibodies (aGai/o/t/z) induced an ectopic partial dorsal axis, with a potency of anti-Gas/olf @ anti-Gai/o/t/z. Numbers of embryos examined are shown above bars. (B) Embryos injected ventrally with 8 ng/blastomere of anti-Gas/olf, anti-Gai/o/t/z, or anti-Gaq/11 antibodies or normal rabbit IgG are shown. Arrows indicate the ectopic dorsal axis induced by anti-Gas/olf and anti-Gai/o/t/z antibody injection. Embryos at tadpole stage 37/38 are shown. (C) Transverse sections of embryos at stage 37/38 which were previously injected with 8 ng/blastomere of anti-Gas/olf or anti-Gai/o/t/z antibodies into two ventral blastomeres are shown. Arrows indicate the ectopic neural tube (2nd nt). No ectopic notochords were observed. Ectopic muscle (m) fused across the midline, as well as unorganized ectopic neural tissues (n), was observed. (D) Injection of anti-Gas/olf or anti-Gai/o/t/z antibodies respecifies the molecular characteristics of VMZs to those of DMZs. VMZs and DMZs were explanted from stage 10–10.5 early gastrula embryos which received injections. RNA from the explants was analyzed by RT-PCR for the presence of chordin, noggin, Xvent1, and EF-1a. EF-1a is a loading control. non, uninjected; aGas/olf, anti-Gas/olf antibody injected; aGai/o/t/z, anti-Gai/o/t/z antibody injected; rIgG, normal rabbit IgG injected. Lanes: E, whole embryos; D, DMZs; V, VMZs; 2, a negative control that contains no RT. chordin and noggin were induced in anti-Gas/olf and anti-Gai/o/t/z antibody-injected marginal zone explants. noggin was induced in anti-Gas/olf but weakly in anti-Gai/o/t/z antibody-injected marginal zone explants.
	FIG. 4. Induction of ectopic axis upon inhibition of Gas does not involve signaling through inhibition of adenylyl cyclase. (A) Peak changes in [cAMP], shown as % ratio normalized by baseline values, are compared among animal caps injected with SQ22536 (200 pmol/blastomere, n 5 13) or MDL12330A (4 pmol/blastomere, n 5 18; 12 pmol/blastomere, n 5 11) and those of the control no-drug-injected animal caps (n 5 19). Injections were made into each of the four blastomeres at the four-cell stage. Assays were done at stage 8–9. Injection of adenylyl cyclase inhibitors SQ22536 (200 pmol/blastomere) or MDL12330A (12 pmol/blastomere) significantly inhibited Gas-mediated activation of adenylyl cyclase upon ligand (arterenol) application in b2AR-overexpressing animal caps (SQ22536, Mann–Whitney U test, *P , 0.001; MDL12330A, Mann–Whitney U test, P , 0.01). Values are expressed as means 6 SEM. (B) Ventral injection of SQ22536 (200 pmol/blastomere) or MDL12330A (12 pmol/blastomere) showed no gross effect on embryonic development. (C) Histogram of frequency of axis duplication induced by ventral injection of different doses of SQ22536 or MDL12330A. These inhibitors showed no effect in inducing an ectopic dorsal axis. Numbers of embryos examined are shown above bars.
	FIG. 5. Inhibition of Gbg subunits induced an ectopic axis. (A) Overexpression of bARK-CT as low as 1 ng/blastomere can induce a partial ectopic dorsal axis. Increasing dose of bARK-CT up to 2 ng/blastomere induced a combined posterior truncation with an ectopic axis formation. Overexpression of bARKNT as a negative control had no effect. Embryos are shown at stage 37/38. (B) Histogram of frequency of axis duplication induced by ventral injections of different doses of bARK-CT, bARKNT, or b-globin. Numbers above the bars indicate numbers of embryos examined. (C) Hematoxylin and eosin staining of transverse section of embryo injected with 1 ng/blastomere bARK-CT RNA. Arrow depicts an induced ectopic neural tube. (D) Injection of bARK-CT, but not bARKNT or b-globin, respecifies the molecular characteristics of VMZs to those of DMZs. RNA from the explants was analyzed by RT-PCR for the presence of chordin, a-actin, Xvent-1, and EF-1a. bARK-CT, bARK-CT RNA injected; bARKNT, bARKNT RNA injected; bglobin, b-globin RNA injected.
	FIG. 6. Gas-coupled b2AR activation elicits IICR that is inhibited by anti-Gas/olf antibodies. (A and B) Normal rabbit IgG- (A) and anti-Gas/olf antibody- (B) injected Xenopus animal caps. Activation of exogenously expressed Gas-coupled b2AR elicited intracellular Ca21 elevation in a small number of cells in control animal caps (A) but not in animal caps injected with anti-Gas/olf antibody (B). Embryos at the two-cell stage were injected sequentially with the calcium indicator fura-2 at 20 mM, an RNA cocktail of Gbg and bAR RNAs, and then normal rabbit IgG or anti-Gas/olf antibody. Animal caps of blastula stage (stage 8–9) embryos were excised and subjected to calcium imaging. Time course of Ca21 changes (F340/F380 ratio) in cells indicated by circles is plotted by the same colors. (a and e) Fura-2 fluorescence images. (b–d and f– h) Ca21 images (F340/F380 ratio) at time points indicated in the plots. The stippled bars show application of 10 mM arterenol. (C) Ca21 elevation by Gas-coupled b2AR activation was inhibited by anti-Gas/olf antibody. Since only a small portion of the total animal cap cells showed Ca21 transients, the number of responding cells was counted. Ca21 elevation in animal caps was divided into four categories according to the number of cells which showed Ca21 transients: 2, no cell; 6, less than 5 cells; 1, 5–10 cells; 11, more than 10 cells. Filled bars, anti-Gas/olf antibody-injected animal caps; open bars, uninjected animal caps; gray bars, normal rabbit IgG-injected animal caps. The distribution pattern of anti-Gas/olf antibody-injected animal caps in these categories was significantly different from that of normal rabbit IgG-injected animal caps (P , 0.05, Mann–Whitney U test) and from that of uninjected animal caps (P , 0.05).
	FIG. 7. Schematic drawing of the signaling pathway for the ventralizing signal. Receptor activation by agonists releases Ga and Gbg subunits as active forms. Active Gas or Gai/o subunits stimulate or inactivate adenylyl cyclase (AC), respectively. Free Gbg dimer activates PLCb. PLCb catalyzes the hydrolysis of PIP2 to produce diacylglycerol and IP3. IP3 in turn binds to IP3R and releases Ca21 from its cytoplasmic storage site, endoplasmic reticulum, to the cytosol. Inhibitory antibodies raised against C-terminal receptor-interacting portions of G protein a subunits block activation of G proteins and therefore cannot distinguish the effect between Ga subunits and bg dimer. To test the function of as subunit, adenylyl cyclase inhibitors, MDL 12330A and SQ22536, were used. For blocking the active bg dimer, bARK was used to titrate out free bg dimers. In this study, we showed that while injection of MDL 12330A and SQ22536 resulted in no effect in the dorsal–ventral fate, injection of anti-Gas/olf or anti-Gai/o/t/z antibodies and bARK-CT into the ventral part of the early Xenopus embryos led to respecification of ventral fate to a dorsal one. Although the potency of each inhibitor differs, the phenotype was similar to that which we previously reported by injection of a functional inhibitory antibody directed against IP3 receptor (anti-IP3R Ab) (Kume et al., 1997b). Arrows, activation; arrowhead with broken line, inhibition; T-shape bar, block.