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