January 1, 2010;
Xwnt8 directly initiates expression of labial Hox genes.
Hox transcription factors play an essential role in patterning the anteroposterior axis during embryogenesis and exhibit a complex array of spatial and temporal patterns of expression. Their earliest onset of expression in vertebrates is during gastrulation in a temporally collinear sequence in the presomitic/ventrolateral mesoderm
, and it is not clear which upstream signal transduction events initiate this expression. Using Xenopus, we present evidence that Xwnt8
is necessary for initiation of this collinear sequence by activating Hox-1 expression in three Hox clusters: hoxd, hoxa, and hoxb. All three labial genes appear to be direct targets of canonical Wnt signaling through Tcf/Lef. In addition, Xwnt8
loss- and gain-of-function leads to indirect regulation of other Hox genes: Hoxb4
, and Hoxc8
. These findings shed new light on the early role of Wnt8
as well as of a proposed WNT gradient in patterning the Xenopus central nervous system (Kiecker and Niehrs  Development 128:4189-4201).
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Figure 1. Expression of Xwnt8 during gastrula and early neurula stages. Embryos were assayed for expression of Xwnt8 by whole-mount in situ hybridization. In each panel, a single embryo is shown. A: Stage 11 embryo, lateral view with dorsal to the left, and a sagittal section of the embryo. Xwnt8 expression is detected in the ventral and lateral marginal zone mesoderm. B: Stage 11.5 embryo, lateral view with dorsal to the left, and a lateral to lateral section. Expression can be found close to the blastopore and in involuted mesoderm. C: Stage 12 embryo, lateral view with dorsal to the left, and a posterior view. Expression of Xwnt8 can be found in presumptive paraxial mesoderm and expression close to the blastopore is further restricted to dorsolateral positions. D: Stage 13 embryo, lateral view with anterior to the left, a posterior view of the embryo, and two transverse sections. In the section on the right top of the panel, Xwnt8 expression in presumptive hindbrain is shown, this corresponds to the anterior-most expression in the lateral view. Expression in mesoderm close to the closing blastopore is shown in the bottom right section of the panel and corresponds to expression shown in posterior view. E: Stage 17 embryo, lateral view with anterior to the left, posterior view, and a transverse section. The anterior ectodermal expression domain, the paraxial expression, and the dorsolateral expression in the mesoderm remain, while a lateral expression domain appears in the ectoderm. In the dorsal-to-ventral section, and in an enlargement on the bottom right of the panel, initiation of Xwnt8 expression in the neural tube can be found.
Figure 2. Expression of Xwnt8, Hoxd1, Hoxb4, and Hoxc6 during gastrulation. A-D: Embryos were analyzed by whole-mount in situ hybridization for expression of Xwnt8 (A), Hoxd1 (B), Hoxb4 (C), and Hoxc6 (D). Embryos are shown, going from left to right through the panels, at stage 11, stage 11.5, stage 12 (vegetal views with dorsal up), and at stage 13 (vegetal views with dorsal up). Xwnt8 expression overlaps with the expression of Hoxd1 in the ventrolateral mesoderm during early gastrulation. At stage 12, the posterior most expression of Xwnt8 becomes restricted to dorsolateral marginal zone, overlapping with the expression domain of Hoxd1. When gastrulation is nearly completed, an overlap in expression of Xwnt8 and Hoxd1 can be observed in presumptive hindbrain, and paraxial mesoderm. Hoxb4 and Xwnt8 show an overlap in their expression patterns during stage 11.5, at stage 12 ectodermal expression of Hoxb4 is initiated in overlapping the dorsolateral Xwnt8 expression domain. During late gastrulation, an overlap in expression of Hoxb4 and Xwnt8 is observed in paraxial mesoderm. Expression of Hoxc6, on the other hand, is initiated after the retraction of the Xwnt8 expression to the dorsolateral domains; therefore, an overlap in expression is only observed there. This overlap is still visible at the end of gastrulation. Likewise for Hoxd1 and Hoxb4, the first ectodermal expression is found in the ectoderm overlying the posterior dorsolateral domains of XWnt8 expression.
Figure 4. Effects of Xwnt8 loss-of-function on expression of Hoxd1, Hoxa1, Hoxb1, Hoxb4, Hoxd4, and Hoxc6, as well as Otx2. Embryos were injected into the animal hemisphere at the two-cell stage with 32 ng of MOXwnt8 per cell and analyzed by whole-mount in situ hybridization. Injected embryos are shown at the bottom of each panel, control embryos are shown on top. Shown are vegetal views with dorsal to the top, except when indicated. A: Expression of Hoxd1 is down-regulated by MOXwnt8 injections, shown are stages 11 (left side of the panel) and stage 12.5 (right side of the panel). B: Expression of Hoxa1 is also down-regulated by the MOXwnt8 at stage 11 (left side of the panel), but it is not visibly affected at stage 13 (right side of the panel). C: Hoxb1 presents a shrinking expression domain upon loss-of-function at stage 13. D: Expression of Hoxb4, shown at stage 11 (left side of the panel) and stage 13 (right side of the panel), is down-regulated in some embryos by Xwnt8 loss-of-function, although in other cases they appear unaffected (see one example of each at st. 13). E: Hoxd4 is down-regulated by the MOXwnt8; shown are stages 11 (left side of the panel) and 13 (right side of the panel). F: Expression of Hoxc6 is up-regulated by Xwnt8 loss-of-function on the dorsal side of the embryo; shown are stages 10.5, where views are lateral with dorsal to the left and the blasopore down (left side of the panel) and 12 (right side of the panel). G: Dorsal to ventral sections of the embryos are shown; the plane of sectioning is depicted by the dotted line in the insets on the bottom left corner. H: Finally, Otx2 expression is increased by MOXwnt8 injections at stage 10.5 (embryos were slightly turned so that the dorsal expression domain could be better seen; the blastopore remains at the bottom).
Figure 5. Effects of Xwnt8 gain-of-function on the expression of Hoxd1, Hoxa1, Hoxb1, Hoxb4, Hoxd4, Hoxc6, as well as Xbra, Xcad3, and Otx2. Embryos were injected at the one-cell stage into the animal hemisphere with 100 pg of CS2-Xwnt8 plasmid and analyzed by whole-mount in situ hybridization. Injected embryos are shown on the bottom of each panel, control embryos on the top. A: Expression of Hoxd1 is ectopically up-regulated in dorsal tissues of injected embryos; shown are stage 10 (left side of the panel) and stage 12.5 (right side of the panel) embryos, the views are dorsal with anterior to the top. B: Hoxa1 expression is up-regulated at stage 11, and the horseshoe pattern closes up in the dorsal midline of the embryo after overexpression of XWnt8 (left side of the panel); at stage 13, there is no evident alteration of expression (right side of the panel); views are vegetal with dorsal to the top. C: pCS2XWnt8 injections lead to a strong ectopic expression of Hoxb1 at both stage 11 (left side of the panel) and stage 13 (middle column of the panel); views are vegetal with dorsal to the top, except for the stage 11 injected embryo, which is seen from the lateral. Dissections of the stage 13 embryos are presented (left side of the panel); embryos were cut along the anterior-to-posterior axis, as depicted by the dotted lines in the insets on the bottom left corner. D: The expression of Hoxb4 is up-regulated at stage 11 and the horseshoe pattern closes up on the dorsal midline of the embryo after Xwnt8 gain-of-function (left side of the panel); expression is not obviously changed at stage 13 (right side of the panel); views are vegetal with dorsal to the top. E: Hoxd4 expression becomes stronger with the XWnt8 gain-of-function, shown are stages 11 (left side of the panel) and 13 (right side of the panel); views are vegetal with dorsal to the top. F: Expression of Hoxc6 is up-regulated dorsally at stage 10 (left side of the panel), and in neuroectoderm of stage 15 embryos (right side of the panel). G: Expression of Otx2 is down-regulated by the overexpression of pCS2XWnt8, shown are stage 10.5 embryos with the blastopore down and the dorsal side to the top. H: Expression of the mesodermal marker Xbra appeared unaltered; shown are stage 11 embryos in vegetal view with dorsal up. I: Finally, expression of the posterior marker Xcad3 is shifted to a more anterior position as a result of the Xwnt8 gain-of-function; shown are embryos at stage 17, dorsal view with anterior up.
Figure 8. Different effects of TVGR (a hormone inducible form of XTcf3) and its subsequent activation on the expression of four Hox genes. Embryos were injected at the two-cell stage with 50 pg of TVGR DNA into each cell; before gastrulation CHX was added to prevent protein synthesis, followed by co-addition of DEX to trigger activation of the hormone inducible system. Embryos were fixed after the standard treatment (see Experimental Procedures section). Each row shows in situ hybridizations with probes for the respective genes noted on the right hand side; each column corresponds to the conditions described on top of it. Injection of TVGR does not significantly alter the normal (NIC) expression pattern of these genes, except for Hoxb1, which shows some ectopic expression. Addition of DEX in injected embryos causes substantial up-regulation of Hoxd1, Hoxb1, Hoxa1 and Hoxc6. Co-addition of CHX and DEX in TVGR injected embryos triggers a massive induction of both Hoxd1 and Hoxa1, as well as induction of Hoxb1; under these conditions, Hoxc6 up-regulation caused by DEX alone on TVGR injected embryos is now abolished. All views are vegetal.