Brewster R et al. (2000), Gli2 functions in FGF signaling during antero-p...

XB-ART-10274

Development 2000 Oct 01;12720:4395-405. doi: 10.1242/dev.127.20.4395.

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Gli2 functions in FGF signaling during antero-posterior patterning.

Brewster R , Mullor JL , Ruiz i Altaba A .

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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.

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Species referenced: Xenopus laevis
Genes referenced: eef1a2 evx1 fgf4 foxa2 gli1 gli2 gli3 gsc msx1 myc sall1 shh smad2 tbx2 tbxt tubb2b

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	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 Gli2C’Æ 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 Gli2C’Æ. (C) Inhibition of Xbra expression (arrow) by Gli2C’Æ 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) Gli2C’Æ 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) Gli2C’Æ (C’Æ) 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.