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The potent mesoderm-inducing factors activin and FGF are present as maternally synthesized proteins in embryos of X. laevis. We show that activin can act on explanted blastomeres to induce at least five different cell states ranging from posterolateral mesoderm to dorsoanterior organizermesoderm. Each state is induced in a narrow dose range bounded by sharp thresholds. By contrast, FGF induces only posterolateral markers and does so over relatively broad dose ranges. FGF can modulate the actions of activin, potentiating them and broadening the threshold-bounded dose windows. Our results indicate that orthogonal gradients of activin and FGF would be sufficient to specify the main elements of the body plan.
Figure 1. Induction by Activin of an Organizer-to-Posterior Spectrum
of Genes Showing Multiple Dose Thresholds
RNAase protection assays of RNA extracted from cell aggregates at
stage 17.5. Single cells had been exposed at blastula stages to a
1.7.fold dilution series of activin. This experiment was performed four
timeswith thesame results. For each experiment, two separate assays
were performed on RNA from the same aggregates, one with keratin
and actin probes (loading control: cytoskeletal actrn), the other with all
other probes together (loading control: EFl-a). Embryo track corresponds
to RNA from three whole stage 17.5 embryos. Genes probed
were:
Epidermal keratin XK81A (Jonas et al., 1989); Xhox3 posteriorenriched
homeobox gene (Rub! i Altaba and Melton, 1989a, 1989b;
Saha and Grainger, 1992); XIHboxG antennapaedia family posterolatera1
specific homeobox (Fritz and De Robertis. 1988; Wright et al.,
1990); Actin, muscle and cytoskeletal (Mohun et al., 1984); Xbra, Xenopus
brachyurygene homolog (Smith et al.,1991); goosecoidorganrzerspecific
homeobox gene (type A, Blumberg et al., 1991; Cho et al.,
1991).
Frgure 2. Whole-Mount In Sttu Hybridization Showrng Expression of
Xbra in Notochord and Posterror Domains
Anterior IS to the left. In srtu hybndrzation with a Xenopus Brachyury
probe was carried out according to Harland (1991).
Figure 3. Induction of Notochord without Coinduction of Muscle
All panels are sections of Stage 42 specimens stained with the antinotochord
sheath antibody MZ15 (Smith and Watt, 1985). (A) shows
a negative control section through an aggregate of untreated cells. (B)
shows a section through an aggregate of cells that were treated as
dispersed cells for 1 hr at blastula stages with 2.5 U/ml activin. A fine
tracery of positive staining is seen throughout the section (though
somewhat less intensely at the center of the section where strucutral
preservation is less good), as is vacuolation characteristicof notochord
differentiation. (C)shows a section through a whole embryo, a glancing
longitudinal section revealing notochord. All panels same scale; scale
bar = 200 Km.
Figure 4. Inductron by FGF of Posteriorly-Expressed Genes Showing
Response Plateaus
RNAase protection assays of RNA extracted from cell aggregates at
stage 17.5. Single cells had been exposed at blastula stages to a
1.7.fold dilution series of basic FGF. Thus experiment was performed
four times with the same results. For each experiment, two separate
assays were performed on RNA from the same aggregates, one with
keratin and actin probes (loading control: cytoskeletal actin), the other
with all other probes together (loading control: EFI-a). Embryo track
corresponds to RNA from three whole embryos. Genes probed were
as in Figure 2. goosecoidexpression was not induced at any FGF dose
(data not shown).
Frgure 5. FGF Modulates Activin Induction Thresholds
Constant concentration of FGF wasadded tovarying activin concentration.
RNAase protection assays of RNA extracted from cell aggregates
at stage 17.5. Single cells had been exposed at blastula stages to a
2-fold dilution series of XTC-MIF (activin) in the presence or absence
of a constant concentration of FGF. This experiment was performed
four trmes wrth the same results
Frgure 6. Dose-Dependent Effects of Activin and FGF and Therr Correspondence to Two Axes of the Mesodermal Elastula Fate Map
(A) Summary of the data shown in Figures l-4 and Table 1. Dashed lines interpolate thresholds at different FGF concentrations assumrng only
that they vary smoothly. (B) Schematic diagram of the Xenopus blastula fate map based on the fate maps of Cooke and Webber (1985) and Keller
(1976). (C) Two-gradient hypothesis for how gradients of activin- and FGF-like activities might specify the fate map. A putative activin-like gradient
has a high point near the prospective dorsal lip, and a putative FGF-like gradient is radially symmetrical about the animal-vegetal axis. It is high
in the upper marginal zone and trails off gradually towards the vegetal pole. Both gradients are in the form of maternal proteins. Sample superimposition
of the separate effects of the two gradients would give rise to the blastula specification map (Dale and Slack, 1987). Interaction between the
gradients takes place during gastrula stages and may require the action of an activin-induced third signal (“dorsalization”; Dale and Slack, 1987).
See text for further explanation.