Witta SE and Sato SM (1997), XIPOU 2 is a potential regulator of Spemann's O...

XB-ART-16815

Development 1997 Mar 01;1246:1179-89. doi: 10.1242/dev.124.6.1179.

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XIPOU 2 is a potential regulator of Spemann's Organizer.

Witta SE , Sato SM .

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XIPOU 2, a member of the class III POU-domain family, is expressed initially at mid-blastula transition (MBT) and during gastrulation in the entire marginal zone mesoderm, including Spemann's Organizer (the Organizer). To identify potential targets of XIPOU 2, the interaction of XIPOU 2 with other genes co-expressed in the Organizer was examined by microinjecting XIPOU 2's mRNA into the lineage of cells that contributes to the Organizer, head mesenchyme and prechordal plate. XIPOU 2 suppresses the expression of a number of dorsal mesoderm-specific genes, including gsc, Xlim-1, Xotx2, noggin and chordin, but not Xnot. As a consequence of the suppression of dorsal mesoderm gene expression, bone morphogenetic factor-4 (Bmp-4), a potent inducer of ventral mesoderm, is activated in the Organizer. Gsc is a potential target of XIPOU 2. XIPOU 2 is capable of binding a class III POU protein binding site (CATTAAT) that is located within the gsc promoter, in the activin-inducible (distal) element. Furthermore, XIPOU 2 suppresses the activation of the gsc promoter by activin signaling. At the neurula and tailbud stages, dorsoanterior structures are affected: embryos displayed micropthalmia and the loss of the first branchial arch, as detected by the expression of pax-6, Xotx2 and en-2. By examining events downstream from the Wnt and chordin pathways, we determined that XIPOU 2, when overexpressed, acts specifically in the Organizer, downstream from GSK-3beta of the Wnt pathway and upstream from chordin. The interference in dorsalizing events caused by XIPOU 2 was rescued by chordin. Thus, in addition to its direct neuralizing ability, in a different context, XIPOU 2 has the potential to antagonize dorsalizing events in the Organizer.

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Species referenced: Xenopus
Genes referenced: bmp4 chrd eef1a2 gsc lhx1 nog not otx2 pou3f4 tbx2 tbxt xpo1

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	Fig. 1. When overexpressed, XlPOU 2 inhibits the expression of a selective group of mesoderm-specific genes as assessed by wholemount in situ hybridization. Two dorsovegetal blastomeres (A-P) or two ventrovegetal blastomeres (Q-T) from embryos at the 8-cell stage were injected with either 200 pg of XlPOU 2 mRNA (B,D,D¢,F,H,I,L,N,P,R,T) or 200 pg of tXlPOU 2 mRNA (A,C,E,G,I,K,M,O,S). The expression of chordin (A,B), BMP-4 (C,D,D¢), gsc (E,F), Xpo (G,H), Xlim-1 (I,J), Xbra (K,L,Q,R), Xnot (M,N), Ef1a (O,P) and PV.1 (S,T) is shown in embryos at stage 10.5 (A-R) or stage 11 (S,T). The schematic shows the orientation of all embryos shown except D¢ (side view). All embryos were cleared in benzyl alcohol/benzoate (2:1), except for those embryos shown in views D¢,M,N,S and T.
	Fig. 2. The detection of region-specific mesoderm markers in embryos at stages 10.5 or 11 by an RT-PCR analysis. Two dorsovegetal (labeled sD or tD) or ventrovegetal (labeled sV or tV), blastomeres of embryos at the 8-cell stage were injected with either 200 pg of XlPOU 2 (designated s)or tXlPOU 2 (designated t) mRNA. E are uninjected embryos. (A-C) Separate studies. All experiments were replicated 2-4 times each. For the experiments shown in B, lanes 6-8, labeled sA (XlPOU 2-injected), tAC (tXLPOU 2-injected), and AC (uninjected), animal cap explants were prepared from injected embryos at stages 8-9, and animal caps were allowed to develop until sibling controls reached stage 10 or stage 11. Total mRNA was prepared from pools of 5 to 10 embryos/injection condition, at stages 10 or 11, and subjected to the RT-PCR analysis as described in Materials and Methods.
	Fig. 3. A gel mobility shift analysis showing that XlPOU 2 binds to the distal and proximal elements of the gsc promoter. The analysis was performed as described in Materials and Methods. P, probe alone; L, unprogrammed lysate; S, lysate programmed with a construct containing the complete XlPOU 2 open reading frame in SP64T; T, lysate programmed with a construct containing a truncated XlPOU 2 (no POU domain) in SP64T. The wild-type and mutant sequences of the distal and proximal elements were previously characterized (Watabe et al., 1995). Mutated bases are underlined. The position of the class III POU protein binding site CATTAAT is boxed in both the distal and proximal elements. The postition of a potential homeobox protein binding site ATTA found in the distal element is also boxed. Lanes 10-12, 14-16 and 22-24 show some non-specific binding of a component in the lysate to the mutant oligomer sequences. The arrows designate the correct position of the specific XlPOU 2/oligomer complex.
	Fig. 4. XlPOU 2 suppresses the activin-induced activation of the gsc promoter. This analysis was performed as described in Materials and Methods. Each experiment was replicated 2-3 times.
	Fig. 5. The overexpression of XlPOU 2 leads to the suppression of the eye and facial structures as evaluated using a whole-mount in situ hybidization technique. One D2.1 blastomere of a 16-cell-stage embryo (A) was injected with 200 pg each of XlPOU 2 and b- galactosidase mRNAs (C, neurula stage; E and G, tailbud stage). Control embryos were injected with 200 pg of tXlPOU 2 mRNA (B and D, neurula stage; F and H, tailbud stage). The expression of pax- 6 (C,D), Xotx-2 (E,F) and en-2 (G,H) is shown. (C) The expression of pax-6 in the central nervous system appears reduced on the injected side because of the plane of the embryo as it was photographed.
	Fig. 6. Interaction of XlPOU 2 with GSK-3b, a component of the Wnt-like dorsalizing pathway. In a control experiment, two ventroanimal blastomeres of a 16-cell-stage embryo were injected with 200 pg of tXlPOU 2 mRNA and 200 pg of b-galactosidase (b- gal) mRNA and two ventrovegetal blastomeres were injected with 1 ng of GSK-3b KM mRNA. Views A and A¢ show a tadpole from this experimental control group; [A, a dorsal perspective showing the secondary axis (red) and A¢, a side view of the same tadpole]. Another group of embryos were injected similarly, but with 200 pg of XlPOU 2 mRNA instead of tXlPOU 2 mRNA (B,B¢, a dorsal and side perspective, respectively, show a tadpole from this experimental group). In other experiments, two ventrovegetal blastomeres from embryos at the 16- cell stage were injected with 200 pg of XlPOU 2 mRNA, 200 pg of b-galactosidase, and 1 ng of GSK- 3b KM mRNA (C,C¢, a dorsal and side perspective, respectively, show a tadpole from this experimental group). Note that the reaction product from lineage tracer is dark red in all three views. The nearly 100% contribution of the injected lineage to the secondary axis in views A and B was readily detected.
	Fig. 7. XlPOU 2 does not disrupt chordin signaling and chordin rescues the phenotype induced by the overexpression of XlPOU 2. Two ventrovegetal blastomeres of embryos at the 16-cell stage were injected with 200 pg of chordin and 200 pg of b-galactosidase mRNAs. (A) A tadpole from this experiment. In another experiment, embryos were injected similarly, with 100 pg of chordin, 200 pg of XlPOU 2, and 200 pg of b-gal mRNAs. (B) A tadpole from this expermental group. The lineage from the injected blastomeres (dark red) contributes heavily to the secondary axis (A,B,B¢, a section from the secondary axis shown in view B). (C-E) Tadpoles from chordin ‘rescue’ experiments. (C) Tadpole from a control group, in which two dorsovegetal blastomeres from embryos at the 16-cell stage were injected with 200 pg each of tXlPOU 2 and b-galactosidase mRNAs. (D) A tadpole from an experimental group similarly injected as in C, but with 200 pg of XlPOU 2 instead of tXlPOU 2. There are severe abnormalities in the head region of these embryos. The most severe phenotype from this experimental group is a ‘headless embryo’. (E) A tadpole from an experimental group that had been ‘rescued’ by injecting 200 pg of chordin mRNA.
	Fig. 8. A proposed model of XlPOU’2s function in the Organizer. (A) The Organizer-specific gene, gsc, is induced by an activin-like mesoderm-inducing signal. Gsc prevents the ventralizing signal, Bmp-4, from being expressed in the Organizer. The secreted molecule, chordin, both a dorsalizing signal and a neural inducer, is induced by gsc. Endogenous XlPOU 2 suppresses (dampens) the activation of gsc by mesoderm induction. (B) When XlPOU 2 is overexpressed, the activation of gsc by the activin-like mesoderm induction signal is severely suppressed. The severe reduction in gsc results in the activation of Bmp-4 in the dorsal lip and the loss of chordin expression. The expression of other Organizer-specific genes, such as noggin and Xlim-1 are greatly reduced, while, Xnot expression is unaltered.