Ault KT et al. (1996), Modulation of Xenopus embryo mesoderm-specific ...

XB-ART-18041

Development 1996 Jul 01;1227:2033-41. doi: 10.1242/dev.122.7.2033.

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Modulation of Xenopus embryo mesoderm-specific gene expression and dorsoanterior patterning by receptors that activate the phosphatidylinositol cycle signal transduction pathway.

Ault KT , Durmowicz G , Galione A , Harger PL , Busa WB .

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A role for the phosphatidylinositol (PI) cycle signal transduction pathway in Xenopus mesoderm induction has been revealed by observations of PI cycle activation coincident with this process, combined with the demonstration that Li+ (a PI cycle inhibitor) blocks this response and hyperdorsalizes mesoderm induction in intact embryos or augments growth factor-mediated induction in animal caps. It has been suggested that spatially restricted PI cycle activity in the marginal zone might modulate (but not, itself, activate) mesoderm induction. To better characterize the ability of PI cycle activity to modulate the pattern of mesoderm-specific gene expression elicited by mesoderm-inducing growth factors we have expressed in the embryo exogenous 5-hydroxytryptamine receptors that activate the PI cycle. In embryos, ventral expression and activation of these receptors during mesoderm induction are without obvious effect, whereas dorsal expression and activation yield dorsoanterior-deficient tadpoles. In animal caps induced with activin, simultaneous activation of exogenous 5-hydroxytryptamine receptors inhibits both convergent extension movements associated with dorsal mesoderm induction and the expression of goosecoid, a dorsal-specific gene, but is without effect on expression of a 149 generic mesodermal marker, Xbra. All of these effects of a 149 PI cycle-stimulating receptor are the opposites of those previously reported for the PI cycle inhibitor, Li+. PI cycle activity thus proves able to modulate the dorsal/ventral character of early mesodermal gene expression elicited by growth factor, suggesting a model for mesodermal patterning.

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Species referenced: Xenopus
Genes referenced: gsc htr2c inhba mrc1 tbxt

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	Fig. 1. Injection of 5-HT1cR mRNA into 4-cell embryos yields functional, PI cycle-stimulating receptors at either the dorsal or ventral midlines by the 64-cell stage. (A) Pseudocolor ratio images of [Ca2+]i in a 64-cell embryo expressing 5-HT1cR at the dorsal and ventral midlines in the marginal zone, before (left) and during (right) treatment with 10 nM 5-HT. In these pseudocolor images of the vegetal hemisphere, blue represents low ratio (low [Ca2+]i) and green and yellow represent higher values of [Ca2+]i. See Materials and Methods for details of embryo preparation and imaging. 70 pg of 5- HT1cR mRNA was injected at the dorsal and ventral midlines in this embryo. (B) Quantitation of the 350/380 nm fluorescence ratios averaged over the two responding regions in the experiment described in Fig. 1A. 5-HT was present in the bathing medium during the interval marked by the horizontal black bar. The dashed and solid traces display the response at the dorsal and ventral injection sites, respectively. Responses similar to those shown here are also observed in embryos bathed in nominally Ca2+-free medium + 1 mM EGTA (not shown), in keeping with an intracellular source for the Ca2+ mobilized by receptor activation.
	Fig. 2. Dorsal (but not ventral) 5-HT1cR stimulation yields grossly dorsoanterior deficient tadpoles. Embryos at the 4-cell stage were microinjected in each of two ventral (top panel) or dorsal (bottom panel) blastomeres at the equator with 38 pg 5-HT1cR mRNA, then treated for 2 hours with 10 nM 5-HT beginning at the 64-cell stage. These tadpoles represent the range of phenotypes typically observed.
	Fig. 3. Stimulation of 5-HT1cR inhibits the animal cap elongation response to both exogenous and endogenous dorsal mesoderminducing factors. Cap elongation is expressed as axial ratio (higher ratio = greater elongation; see Materials and Methods). (A) 4-cell stage embryos were microinjected in the animal pole region of each blastomere with 38 pg 5-HT1cR mRNA. At the 512-cell stage animal pole tissue free of marginal zone was excised and treated with the indicated ligands (100 nM 5-HT, 100 nM mianserin, and/or 50 ng/ml activin A). Error bars denote ±s.e.m. (B) 4-cell stage embryos were injected in either the two dorsal (D, control) or ventral (V) blastomeres with 38 pg 5-HT1cR mRNA, or were UV-irradiated (UV) at the 1-cell stage as described in Materials and Methods. At the 64- cell stage D and V embryos were treated with 10 nM 5-HT for 1.5 hours. Caps containing some marginal zone tissue were excised after mid-blastula transition and incubated in MMR + 100 nM mianserin.
	Fig. 4. 5-HT1cR stimulation specifically inhibits the induction of goosecoid expression by activin in animal caps. Caps were prepared at the 1024-cell stage from embryos injected in the animal pole region of each blastomere at the 4-cell stage with 38 pg 5-HT1cR mRNA. Immediately after excision, experimental caps were treated with 50 ng/ml activin plus either 100 nM mianserin (open squares) or 100 nM 5-HT (filled squares) for 1.5 hours. Control caps (not treated with any ligands) were prepared from embryos either injected as above with 5- HT1cR mRNA (filled circles) or from embryos receiving no mRNA (open circles). mRNA amounts are expressed as the ratio of goosecoid (A) or Xbra (B) band intensity to that of EF-1a for the same time point and group of caps, determined simultaneously via duplex RT-PCR (see Methods). The only statistically significant difference between activin-induced caps ± 5-HT is the 2 hour goosecoid time point (P<0.05, Student’s one-tailed t-test for paired data). Symbols and error bars represent the means ±s.e.m. for 3 (control) or 5 (experimental) independent experiments. C is a representative autoradiogram of the PCR data from one experiment; some non-systematic variation in the very low basal levels of Xbra are occasionally observed (first three lanes), but this is not a consistent result. Abbreviations: 0, 2 hours, 6 hours, 15 hours, time at which mRNA was extracted (relative to initiation of ligand treatment at t = 0); A, caps treated with activin + mianserin; 5+A, caps treated with activin + 5-HT; PO, primers only lane (no template present in the PCR reaction).
	Fig. 5. Models for graded activation of the PI cycle during mesoderm induction. Arrow length and direction indicate the relative magnitude and sign, respectively, of the effect of an agent on PI cycle activity (up arrows = stimulation; down arrows = inhibition). Stippling indicates the resulting level of PI cycle activity (darker stippling = higher activity), whereas hatching indicates inhibition. For simplicity, only the marginal zone is considered here. In each figure, the prospective ventral midline is on the left. (A) A single PI cycle stimulator, with highest activity in the ventral hemisphere, directly elicits a gradient of PI cycle activity along the dorsal-ventral axis. (B) The effects of two or more PI cycle stimulators sum to yield a dorsal-ventral gradient. At least one of these agents must have highest activity in the ventral hemisphere, as in A, but this model differs from A in that the magnitude of the effect of any one stimulating agent need not, by itself, be sufficient to raise PI cycle activity to levels that are inhibitory for dorsal-most induction. (C) The effect of a single PI cycle stimulator (which need not be graded in activity) is counteracted in the dorsal hemisphere by a localized inhibitor of PI cycle activity.