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Figure 4. Xlefty Acts at a Distance to Inhibit Endogenous Nodal- and Wnt-Responsive Gene Expression.
(A) Xlefty RNA (100 pg) was injected into a dorsal left cell of the top tier of a pigmented 32-cell embryo. Coinjection of a lineage tracer confirmed targeting (our unpublished data). A is animal pole, Vg is vegetal pole, D is dorsal, and V is ventral. (B–G) Injected embryos were fixed at stage 10.25–10.5 and examined by in situ hybridization so that the endogenous expression of (B and E) Xbra, (C and F) Xnr3, and (D and G) gsc could be assessed. Vegetal views, with dorsal at the top. Arrowheads indicate the dorsal lip. (B–D) Uninjected embryos displayed normal expression of Xbra ([B], n = 20/20), Xnr3 ([C], n = 19/20), and gsc ([D], n = 10/10). In contrast, injection of Xlefty into the animal cap severely reduced or eliminated the endogenous expression of Xbra ([E], n = 20/20 reduced), Xnr3 ([F], n = 11/20 absent; 9/20 reduced), and gsc ([G], n = 9/10 absent; 1/10 reduced). These results indicate that ectopic Xlefty expression in an ectoderm lineage (green, [A]) can inhibit endogenous Nodal- and Wnt-responsive gene expression in distant mesoderm lineages (red and yellow, [A]).
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Figure S3. Post-Gastrula Xlefty-Deficient Exogastrulae Exhibit a
Reversal of the A/P Axis and Normally Patterned Mesoderm and
Endoderm
The expression of gsc and Xbra, markers of the prechordal plate
and notochord, respectively, and of Xcaudal2 (Xcad2) [S9], a marker
of posterior development, indicates a reversed A/P axis and normal
dorsal patterning in the Xlefty-deficient exogastrulae. In addition to
Xbra, the expression of Xenopus sonic hedgehog (Xshh) [S10] and
myoD [S11], markers of the axial and paraxial mesoderm, respectively,
indicates a normal complement of relatively normally patterned
mesoderm in the exogastrulae. The expression of edd indicates
a normal complement of endoderm in the exogastrulae. Left
and right panels show uninjected and anti-Xlefty morpholinoinjected
(MO) albino embryos, respectively. gsc and Xbra were examined
at stage 13/14 (dorsal views, with anterior to the left). All
other markers were examined at stage 25/26 (left lateral views, with
dorsal to the top and anterior to the left). Fifteen to twenty embryos
were examined for each marker by whole-mount in situ hybridization.
Previously, exogastrulae have been used for studying vertical versus planar neural induction, with the assumption that in exogas trulae the mesoderm and ectoderm are never vertically apposed because involution does not occur [S12]. However, the validity of this assumption is still controversial [S13, S14]. Because the Xlefty- deficient exogastrulae undergo some involution (Figure S1), transient vertical apposition of the mesoderm and ectoderm probably occurs in these embryos and renders them not useful for resolving this controversy regarding neural induction. |
