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Fig. 3. Effect of the inhibition of PI3K signalling on the in vivo
expression of early patterning genes. (A-K) Embryos were injected
equatorially at the four-cell stage in one or two blastomeres with
mRNA for NLS-b-Gal alone (A,D,F,H,J), with ã p85
(0.5 ng/blastomere; B,E,G,I,K), or with ã p85 (0.5 ng/blastomere)
and p110caax (0.5 ng/blastomere, C), and cultured until stage
10.5-11 before fixing and whole-mount in situ hybridisation with the
indicated probe (top right of each part). (L) RT-PCR analysis of the
expression of early trunk mesodermal markers in stage 10.5 whole
embryos (WE) treated between stage 8.5 and 10.5 with 100 mM of
LY294002 (LY). (M) RT-PCR analysis of the expression of early
mesodermal and endodermal markers in stage 10.5 whole embryos
(WE) treated between stage 8.5 and 10.5 with 100 mM of LY294002
(LY). (N) RT-PCR analysis of the expression of Xbra and Siamois
(Sia) at stage 10 in control embryos or embryos incubated in
LY294002 (LY; 25 mM) between the four-cell stage and stage 10.
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Fig. 1. Inhibition of PI3K signalling prevents gastrulation and axial development. (A,B,D) External appearance at the equivalent of the tailbud stage of WT embryos (A); embryos radially co-injected at the four-cell stage with mRNA for ∆p85 (1 ng/blastomere) and NLS-β- Gal (B); or embryos treated with 80-100μM LY2941002 (D, right embryo, side view; left embryo, vegetal view). The majority of treated embryos have no apparent axial or head structures (severe phenotype, left embryos), while a minority of embryos showed reduced axial structures and a cement gland (arrow) but no eye anlage or hatching gland (mild phenotype, right embryos).
(C) Rescue of embryos radially injected with ∆p85 mRNA
(1 ng/blastomere) by co-injection with activated p110α (p110caax, 1 ng/blastomere). In C, trunk and head but not tail development is rescued. (E-G) Vegetal views at the late gastrula stage (st. 12.5) of a control embryo (E), an embryo incubated in 80μM LY294002 between stages 9 and 12.5 (F) and an embryo incubated in 30 μM LY294002 between the two-cell stage and stage 12.5 (G). Gastrulation in embryos F and G has been severely impaired. This phenotype was fully penetrant.
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Fig. 2. Reduction of axial
mesoderm differentiation in
the absence of PI3K
signalling. Whole-mount
immunohistochemistry at the
tailbud stage with monoclonal
antibodies against muscle
(12/101; A-C) or notochord
(MZ15; D-F) on control
embryos (A,D), and on
embryos co-injected in the
equatorial region of four-cell
embryos with mRNAs for
∆p85 (1ng/blastomere) and
NLS-β-Gal (B, injection into
one side of the embryo only; E, radial injection). In B,E, visualisation of the β-gal positive cells (blue X-gal staining) reveals that the inhibition of muscle and notochord development is restricted to the cells that have received the exogenous mRNA. (C,F) Embryos treated with 100 μM LY2941002 from stage 9 to stage 12.5.
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Fig. 4. PI3K signalling is required for
activin- and FGF-mediated mesoderm
induction in animal caps. (A-C) Animal
caps from uninjected embryos (A,B) or
embryos injected at the two-cell stage
with ∆p85 mRNA (1 ng/blastomere, C)
were explanted around the mid-blastula
stage and cultured in the absence (A) or
presence of 4 units/ml of activin (B,C)
until the equivalent of stage 16. ∆p85
prevents the activin-induced elongation
of the caps. (D) Animal caps from
embryos uninjected or injected at the
two-cell stage with mRNA for ∆p85
(1ng/blastomere, animal injection) were
excised at stage 8.5, treated with activin
(4 units/ml) alone or in combination
with LY294002 (LY, 100 μM), frozen at
stage 10 and analysed by RT-PCR for
the expression of Xbra, Mix.1 and
goosecoid (gsc). (E) Animal caps from
uninjected embryos or embryos injected,
when indicated, at the two-cell stage
with mRNA for ∆p85 (1 ng/blastomere,
animal injection) were excised at stage
8, treated when indicated with FGF2 (50
ng/ml) alone or in combination with
LY294002 (100 μM) or wortmannin (10
and 100 nM), frozen at stage 10 and
analysed by RT-PCR for the expression
of Xbra. (F) Animal caps from control embryos or from embryos injected with mRNA for ∆p85 (250 pg/blastomere), p110caax (125 pg- blastomere), or a combination of both, were explanted during the blastula stages and cultured when indicated in 50 ng/ml FGF2 until the early gastrula stage and processed for RT-PCR with Xbra primers. (G) Animal caps were excised at stage 8, treated with FGF2 (50 ng/ml) alone or in combination with LY294002 (100 μM) or cycloheximide (CHX, 10 μg/ml) as indicated, and frozen at stage 10 for RT-PCR analysis of Xbra expression (top panels). In D-G, the ubiquitously expressed FGF receptor 1 gene (FGFR) is used as a loading control.
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Fig. 5. Position of PI3K in the FGF signalling cascade. (A) Embryos uninjected or injected at the two cell-stage animally with the indicated combination of 40 pg/blastomere of Ras (v-ras) mRNA and/or 1 ng of ∆p85 mRNA were incubated with cycloheximide (CHX, 10 μg/ml) from stage 7 onwards where stated. Animal caps were excised at stage 8 and frozen at stage 10 for RT-PCR analysis of Xbra expression.
(B) Embryos uninjected or injected animally at the two-cell-stage with 40 pg/blastomere of Ras mRNA were incubated when indicated with LY294002 (25 μM) from the four-cell stage onwards and/or with cycloheximide (CHX, 10μg/ml) from stage 7 onwards. Analysis of Xbra expression at stage 10 was performed as in A. (C) Effect of LY294002 treatment on the expression of Xbra at stage 10 in animal caps injected at the two-cell stage with MEK1S217E/S221E mRNA (125 pg/blastomere) and treated with LY294002 in the same way as in panel B. (D) Two models of action of PI3K in the FGF pathway. The identity of the molecules blocked by ∆p85 and LY294002 (red) is shown. (E) Western blot analysis of the effect of LY294002 on the activation by phosphorylation of ERK in response to FGF signalling. Animal caps from control embryos or embryos preincubated in 100 μM LY294002 for 2 hours were explanted at stage 9 and cultured for 20 minutes in Modified Barth Saline (MBS) or MBS + 100μM LY294002. ERK is present in equal amounts in fully grown stage VI and progesterone-matured oocytes, but is only activated in the latter. The stage VI and mature oocytes lanes demonstrate the specificity of the anti-activated ERK antibody used. The low level of activation of ERK in control animal caps may be due to the wounding of the cells during the explantation (LaBonne and Whitman, 1997). P-ERK, activated ERK; ERK, total ERK.
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Fig. 6. Activated ERK and p110 synergise during mesoderm induction in animal caps. (A-D) Whole-mount photography of stage 37/38 animal caps explanted at stage 9 from control embryos (A), embryos injected with 250 pg of mRNA for p110caax, a membrane- targeted form of the p110α class 1A PI3K (B), embryos injected with 200 pg of mRNA for Xp42D324, an activated form of ERK (C) or embryos co-injected with both mRNAs (D). (E,F) Histological staining of sectioned animal caps at stage 37/38. (E is a control cap; the cap in F expresses activated ERK and p110α). Τhe presence of a large fluid-filled cavity lined with mesothelial cells (arrows) is characteristic of ventral mesoderm differentiation (Smith, 1993). This analysis did not reveal significant difference between the mesodermal types induced by the different mRNA injections.
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