Nishinakamura R et al. (1999), Activation of Stat3 by cytokine receptor gp130 ...

XB-ART-11625

Dev Biol 1999 Dec 15;2162:481-90. doi: 10.1006/dbio.1999.9518.

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Activation of Stat3 by cytokine receptor gp130 ventralizes Xenopus embryos independent of BMP-4.

Nishinakamura R , Matsumoto Y , Matsuda T , Ariizumi T , Heike T , Asashima M , Yokota T .

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Stat3 is one of the main signaling components of cytokine receptors, including gp130. Here we show that activation of cytokine receptor gp130 resulted in a dramatic ventralization of Xenopus embryos and that the ventralization correlated well with Stat3 activation potential of the receptor. This finding led to identification of Xenopus Stat3 (Xstat3), which showed a 95% homology to its murine and human counterparts, at the amino acid level, and was expressed from the one-cell stage throughout development. The mechanism of gp130/XStat3-mediated ventralization proved to be independent of BMP-4. gp130/Xstat3 stimulation inhibited Smad2-induced ectopic axis formation in embryos and Smad2-dependent luciferase activity. A dominant-negative Stat3, in contrast, dorsalized Xenopus embryos, resulting in ectopic axis formation. We propose that Stat3-mediated signaling has the capacity to modify dorsoventral patterning in the early development of Xenopus.

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Species referenced: Xenopus
Genes referenced: bmp2 bmp4 cntn1 il6st lif nog smad1 smad10 smad2 smad4 stat3 stat3.2 ventx2.2 wnt8a

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	FIG. 1. The ventralizing effects of gp130 are closely linked to its Stat3-binding sites. (A) Chimeric receptor constructs. (Left) a-gp130 and b-gp130. Extracellular regions of human GM-CSF receptors (a and b subunits) were fused to transmembrane and intracellular regions of mouse gp130. (Middle) F2 mutants. The C-terminal regions of the chimeric receptors were truncated up to the 133rd amino acid and tyrosine 118 was replaced by phenylalanine. These mutants lack the Ras–MAP kinase pathway, but retain Stat3 signaling. The amino acid numbering is counted from the starting point of the intracellular region of gp130. (Right) F3 mutants. The C-terminal regions of the chimeric receptors were truncated up to the 133rd amino acid and tyrosine 126 was replaced by phenylalanine. These mutants lack Stat3 signaling but retain the Ras–MAP kinase pathway. (B) (Upper left) RNA from a-gp130, b-gp130, and GM-CSF ligand (20 pg each) was injected into equatorial regions of two dorsal blastomeres of 4-cell-stage embryos. The average dorsoanterior index (DAI) was 1.78 6 0.81 (n 5 50). (Upper right) Injection of RNA (20 pg each) from GM-CSF and F2 mutants (a-F2 and b-F2). DAI 1.92 6 1.21 (n 5 23). (Lower left) Injection of RNA (20 pg each) from GM-CSF and F3 mutants (a-F3 and b-F3). DAI 4.42 60.76 (n524). (Lower right) Uninjected controls.
	FIG. 2. Histological examination of embryos injected with gp130. (A) A sagittal midline section of an uninjected embryo at tadpole stage (stage 42). (B) A sagittal midline section of an embryo injected with 20 pg each of RNA from a-gp130, b-gp130, and GM-CSF into two dorsal blastomeres at the 4-cell stage (stage 42). (C) A transverse section at the level of the bar indicated in A of an uninjected embryo (stage 42). (D) A transverse section at the level of the bar indicated in B of an embryo which was injected with the gp130 mixture and showed a phenotype similar to that of B (stage 42). bl, blood island; cg, cement gland; ev, ear vesicle; fb, forebrain, gu, gut; hb, hindbrain; no, notochord; sc, spinal cord; so, somite; yo, yolk. Scale bar, 200 mm.
	FIG. 3. Whole-mount in situ hybridization of embryos upon gp130 stimulation. RNA from a-gp130, b-gp130, and GM-CSF ligand (20 pg each) was injected into equatorial regions of all blastomeres of 4-cell-stage albino embryos and the resulting embryos were analyzed by whole-mount in situ hybridization at the gastrula stage (right). Embryos are shown in vegetal view with the dorsal side up. Left, uninjected controls.
	FIG. 4. Xstat3 is present throughout early development and activated upon gp130 stimulation. (A) Xstat3 protein is present from the one-cell stage throughout early development. Proteins from one embryo equivalent were blotted using an anti-Stat3 antibody. (B) Xstat3 is activated upon gp130 stimulation. APRF-luc was co-injected with the indicated RNA into two dorsal blastomeres of 4-cell-stage embryos. Five embryos for each sample were harvested at stage 10 and subjected to luciferase assay. Luciferase activity from embryos injected with APRF-luc alone was set as baseline activity. gp130, GM-CSF1a-gp1301b-gp130; F2, GM-CSF1a-F21b-F2; F3, GM-CSF1a-F31b-F3 (20 pg each); BMP-4 (100 pg); Xwnt-8 (2 pg); and activin (20 pg). (C) Xstat3 is not activated by BMP-4, BMP-2/4, or Smad1/4. Luciferase assay was done as in (B). BMP-4 (100 pg), BMP-2/4 (BMP-2 100 pg, BMP-4 100 pg), and Smad1/4 (Smad1 100 pg, Smad4 100 pg).
	FIG. 5. The relationship of gp130/Xstat3 signaling with BMP-4 and Smad2. (A) gp130 stimulation inhibited axis duplication induced by noggin, a dominant-negative BMP-4 receptor (DNBR), and Smad2. Indicated RNA was injected with 60 pg of EF-1a RNA (2) or RNA (20 pg each) from GM-CSF, a-gp130, and b-gp130 (1) into two ventral blastomeres of 4-cell-stage embryos. Numbers of embryos examined are beside the bars. Doses of RNA used were as follows: noggin (2 pg), the dominant-negative BMP-4 receptor (100 pg), and Smad2 (500 pg). (B) Xvent2-luc (BMP-4-responsive element) was co-injected with the indicated RNA into two ventral blastomeres of 4-cell-stage embryos. DNBR (100 pg), GM-CSF1agp1301b- gp130 (gp130) (20 pg each). Luciferase activity from embryos injected with DNBR was set as baseline activity. (C) DE-luc (activin-responsive element) was co-injected with indicated RNA into one ventral animal (A) or ventral vegetal (V) blastomere of 8-cell-stage embryos. Activity in the ventral animal blastomere was set as baseline activity. Smad2 (300 pg), GM-CSF1a-F21b-F2 (F2) (20 pg). Experiments were repeated at least three times and one representative experiment is shown in each graph. Each experiment was carried out using eggs laid by a single female.
	FIG. 6. Xstat3EnR induced ectopic axis formation. (A) Structure of Xstat3 and Xstat3EnR. TAD, transactivation domain; Y, tyrosine residue of which phosphorylation is required for dimerization. (B) Xstat3EnR has dominant-negative activity. Xstat3EnR inhibited LIF-induced Stat3 activation measured by APRF-luc in BOSC cell lines, while Xstat3EnR containing mutations in its DNA binding domain did not. 2 mg of the indicated DNA driven by the CAG promoter was transfected into BOSC cell lines along with APRF construct. 24 h after transfection, cells were split and cultured with or without LIF. Cells were harvested after 24 h of culture and assayed for luciferase activity. Each bar represents data from triplet samples. Mock, CAG vector with no inserts was transfected as a control; closed bars, without LIF; open bars, in the presence of LIF. Mock transfection without LIF was set as baseline activity. (C) Xstat3EnR (30 pg), but not the mutated form (300 pg), induced an ectopic axis formation, which was inhibited by co-injection of Xstat3 (500 pg), but not by GM-CSF RNA (500 pg). Numbers of embryos examined are beside the bars. (D) (Upper left) Xstat3EnR RNA (100 pg) induced ectopic axes in embryos. (Upper right) Injection of Xstat3EnR (30 pg) also resulted in axis duplication. (Lower left) Xstat3EnR (30 pg) was co-injected with GM-CSF RNA (500 pg). (Lower right) Xstat3EnR (30 pg) was co-injected with Xstat3 RNA (500 pg), resulting in inhibition of ectopic axis formation.
	FIG. 7. Xstat3EnR dorsalized ventral marginal zones. RNA of Xstat3EnR (100 pg) or mutated Xstat3EnR (100 pg) was injected into equatorial regions of all blastomeres of 4-cell-stage embryos. Dorsal marginal zone (DMZ) and ventral marginal zone (VMZ) were isolated at stage 10 and cultured until sibling embryos reached stage 11 (top) or stage 28 (bottom). RNA from explants was analyzed by RT-PCR. D, DMZ; V, VMZ; E, whole embryo. (2) Negative control that contained no reverse transcriptase.