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Patterning along the anteroposterior (A-P) axis involves the interplay of secreted and transcription factors that specify cell fates in the mesoderm and neuroectoderm. While FGF and homeodomain proteins have been shown to play different roles in posterior specification, the network coordinating their effects remains elusive. Here we have analyzed the function of Gli zinc-finger proteins in mesodermal A-P patterning. We find that Gli2 is sufficient to induce ventroposterior development, functioning in the FGF-brachyury regulatory loop. Gli2 directly induces brachyury, a gene required and sufficient for mesodermal development, and Gli2 is in turn induced by FGF signaling. Moreover, the homeobox gene Xhox3, a critical determinant of posterior development, is also directly regulated by Gli2. Gli3, but not Gli1, has an activity similar to that of Gli2 and is expressed in ventroposterior mesoderm after Gli2. These findings uncover a novel function of Gli proteins, previously only known to mediate hedgehog signals, in the maintenance and patterning of the embryonic mesoderm. More generally, our results suggest a molecular basis for an integration of FGF and hedgehog inputs in Gli-expressing cells that respond to these signals.
Fig. 1. Expression of SalB, Gli2 and Gli3
in mesoderm. (A) Endogenous
expression of SalB in the marginal zone
of early gastrula (stage ~10, left), and in
the notochord and circumblastoporal
region of late gastrula-early neurula
(stage ~13, right) embryos. Weak
expression is also present in the posterior
neural plate. The embryo to the left
shows a vegetal view and the one to the
right shows a dorsoposterior view.
(B) Diagram of the structure of the Xsal1
(Hollemann et al., 1996), SALL1
(Kohlhase et al., 1996) and SalB
predicted proteins. Zinc fingers are
denoted by vertical ellipses. Percent
identities are given between homologous
regions limited by broken lines. Note the
lack of the last two sets of two fingers in
SalB and the premature termination (*).
The percent homology given for the
region C-terminal to the stop codon (*) corresponds to the hypothetical ORF that would be in frame in a nonspliced version. (C) RT-PCR
analyses localizing the expression of Gli2 to the ventral equatorial region of late blastulae/early gastrulae, like Msx1 and unlike goosecoid
(Gsc). Expression of the housekeeping gene EF1a is used as control. (D,E) Endogenous expression (arrows) of Gli2 (D) and Gli3 (E) in the
circumblastoporal region of late gastrula (stage ~12.5-13) embryos. (E) A filleted embryo viewed from the inside, localizing Gli3-expressing
cells to the mesodermal layer (arrows). bp, blastopore; ect, ectoderm; end, endoderm; mes, mesoderm. In all cases anterior is towards the top
except in A, left, which shows a vegetal view.
Fig. 2. Gli2 induces mesodermal and neuronal differentiation.
(A,B) Ectopic expression of Xbra in a Gli2-injected gastrula (stage
~12.5-13) embryo (A), but not in Gli1-injected sibling embryos (B).
Double-labeling with anti-Myc antibodies (A,B) reveals the position
of cells expressing the injected Myc-tagged Gli proteins. The inset in
(A) shows two Myc-Gli expressing cells in a histological section, of
which only one also expresses Xbra (arrow). (C) Ectopic expression
of SalB in a protrusion (arrows) induced by injected Gli2 in a stage
~12.5-13 embryo. (D) Induction of ectopic N-tubulin (Ntub)
expression in Gli2-injected embryos at neurula stages (stage ~15).
Note the protrusions and the separation of Myc+/N-tubulin- cells
from Myc+/N-tubulin+ cells (inset). (E,F) Induction of ectopic Gli2
(E) and SalB (F) expression in late gastrula embryos by injected
Xbra. In each part, arrows point to sites of ectopic gene expression.
The panels show dorsal views in which anterior is towards the top,
except in (E,F) in which it is towards the left. Myc labeling is brownred
and in situ hybridization signal is blue.
Fig. 3. Gli2 and Gli3 induce ectopic tail development.
(A-D) Expression of injected lacZ alone in epidermis (A) does not
affect the morphology of the resulting tadpole (stage ~32), whereas
co-injection of lacZ (as a lineage marker) and Gli2 RNAs result in
the induction of tails (B-D, arrows) in stage ~34 embryos. b-gal+
cells accumulate at the tail tips. The inset in C shows a histological
section of an induced tail in a stage ~32 embryo showing the
presence of labeled cells in both mesoderm (mes, arrow) and
overlying ectoderm. The embryo in (D) received two injections into
each cell at the two-cell stage and developed bilateral ectopic tails
(arrows). (E) Gli2-injected embryos show ectopic tails (arrows) that
lack Shh+ cells and thus lack notochord, hypochord or floor plate
cells. (F) Ectopic tails induced by Gli2 rarely contain myoD+ cells,
indicating that somitic tissue is not often included. (G) Xen1+ neural
differentiation is detected in Gli2-induced ectopic tails (arrow). (H)
Ectopic tails (arrow) induced by Gli3 or Gli2 (not shown) lack floor
plate (fp) or endodermal cells as judged by the absence of HNF-3b
expression. The endogenous floor plate is labeled. In all panels,
arrows show the position of the ectopic tails. Whole mounts show
cleared embryos in F,G. Anterior is towards the left except in (H).
All embryos are shown at tadpole stages (stages ~32-34).
Fig. 4. Effects of repressor and activators on Xbra expression and
involvement of Gli2 in the FGF-brachyury pathway. (A) Expression
of Gli2Câà in the marginal zone of a stage ~10 embryo inhibits
endogenous Xbra expression. Note that the arc of Myc label
(brown) coincides with the gap (arrow) in Xbra expression
(purple). (B) Posterior deficiencies in embryos (stage ~32)
injected with lacZ plus Gli2CâÃ. (C) Inhibition of Xbra
expression (arrow) by Gli2Câà in a stage ~10 embryo is not
rescued by co-expression of activated activin type I receptor
(RIB*). (D) Inhibition is also not rescued by co-expression of
activated Smad2-lacZ fusion (S2) in a stage ~10 embryo. Note the
light-blue b-gal product overlapping the Myc label in the Xbra gap
(arrow). (E) Gli2Câà inhibits ectopic Xbra expression (arrow)
induced by co-injected eFGF in a cell-autonomous manner in the
animal cap of a stage ~10 embryo. Note the light-purple label
around Myc-labeled (brown) cells in the animal cap. (F) Full-length
Gli2 inhibits Xbra (arrows and bracket) by co-expressed eFGF in a
stage ~11 embryo. Note the gap (bracket) between Myc-expressing
and Xbra-expressing cells, indicating non-cell-autonomous
repression. (G) Inhibition of endogenous Xbra expression at a
distance from cells expressing the activator VP16Zic2 protein. The
bracket shows the region of non-cell-autonomous repression of
Xbra in between cells maintaining Xbra and cells inheriting the
injected Myc-labeled material (arrows). (H) Gli2Câà (CâÃ) inhibits
the non-cell-autonomous repression of Xbra by co-expressed
VP16Zic2. The arrow indicates the junction of Xbra expression and
Myc label, as in A. Embryos in (A,C,G,H) show vegetal views.
(B) shows a lateral view with anterior towards the left (top and
bottom right embryos) or towards the right (bottom left embryo).
(D,F) show views of the marginal zone. (E) shows a view of the
animal pole.
Fig. 5. Gli2 and brachyury induce
mesoderm formation and elongation in
isolated animal caps. (A-E) RT-PCR
analyses of animal cap explants injected
and treated in various ways. The stage is
given and the injected RNAs or treatments
are shown above each column. The name
of the tested genes is given to the left of
each row at the corresponding position.
Note that SalB shows three specific
products in whole embryos but only one
in animal caps. (F) Morphological
appearance of uninjected, Gli2-, Xbraand
eFGF-injected albino animal caps
cultured to the equivalent of stage 14.
Note the pronounced elongation of caps
injected with Gli2 or Xbra. Two
representative caps are shown in each
case.
Fig. 6. Gli2 function requires FGF signaling. (A) Induction of
ectopic tails (arrows) in tadpoles after injection of Gli2 plus
lacZ. (B) Expression of XFD in posterior mesoderm antagonized
proper posterior development (arrow; Amaya et al., 1991).
(C) XFD inhibits ectopic tail induction by co-expressed Gli2.
Each panel shows lateral images of two tadpoles at stages ~32-
34 with similar phenotypes. Anterior is towards the left.
