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Development
2006 Sep 01;13318:3709-22. doi: 10.1242/dev.02516.
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Hex acts with beta-catenin to regulate anteroposterior patterning via a Groucho-related co-repressor and Nodal.
Zamparini AL
,
Watts T
,
Gardner CE
,
Tomlinson SR
,
Johnston GI
,
Brickman JM
.
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In Xenopus, the establishment of the anteroposterior axis involves two key signalling pathways, canonical Wnt and Nodal-related TGFbeta. There are also a number of transcription factors that feedback upon these pathways. The homeodomain protein Hex, an early marker of anterior positional information, acts as a transcriptional repressor, suppressing induction and propagation of the Spemman organiser while specifying anterior identity. We show that Hex promotes anterior identity by amplifying the activity of canonical Wnt signalling. Hex exerts this activity by inhibiting the expression of Tle4, a member of the Groucho family of transcriptional co-repressors that we identified as a Hex target in embryonic stem (ES) cells and Xenopus embryos. This Hex-mediated enhancement of Wnt signalling results in the upregulation of the Nieuwkoop centre genes Siamois and Xnr3, and the subsequent increased expression of the anterior endodermal marker Cerberus and other mesendodermal genes downstream of Wnt signalling. We also identified Nodal as a Hex target in ES cells. We demonstrate that in Xenopus, the Nodal-related genes Xnr1 and Xnr2, but not Xnr5 and Xnr6, are regulated directly by Hex. The identification of Nodal-related genes as Hex targets explains the ability of Hex to suppress induction and propagation of the organiser. Together, these results support a model in which Hex acts early in development to reinforce a Wnt-mediated, Nieuwkoop-like signal to induce anterior endoderm, and later in this tissue to block further propagation of Nodal-related signals. The ability of Hex to regulate the same targets in both Xenopus and mouse implies this model is conserved.
Fig. 1. Hex anteriorises the phenotypes induced by β-catenin and regulates its downstream targets. (A) Axis duplication phenotypes induced by Hex and β-catenin. Embryos were injected with 500 pg Hex,β -catenin, or both RNAs into a single-ventralblastomere at the four-cell stage. In situ hybridisation was performed for the anterior neural marker BF1 at stage 35. The inset and arrowheads indicate a small ventral outgrowth produced by Hex injection. (B) Phenotypes induced by Hex andβ -catenin in ventral marginal zone explants. Embryos were injected with the indicated RNA into both blastomeres at the two-cell stage, cultured to gastrulation, VMZ explants dissected and cultured until staging control embryos reached stage 35. BF1 in situ hybridisation was performed to highlight the phenotypes obtained. Hex RNA was injected at 500 pg. (C,D) Real-time RT-PCR analysis of Siamois and Xnr3 expression in VMZ explants analysed at stage 10.5. Embryos were injected as in B with the indicated RNA. Values were normalised to the expression level of Odc and the relative change in gene expression for the genes analysed was calculated by dividing the values from injected samples by the values from the uninjected. Data are based on three independent experiments. (E) Cell automonous induction of β-catenin targets Siamois and Xnr3. RNA was injected into a single blastomere at the four-cell stage with the indicated RNA alongside nucGFP RNA. Dorsal injections are indicated with `D' where either 250 pg Hex or 100 pg Hex-λVP2 were used. Ventral injections are indicated with `V' where 500 pg Hex and 500 pg β-catenin were used. Embryos were processed by double in situ hybridisation and stained for both nucGFP to indicate the injected cells (light blue) and Xnr3 (dark blue). The schematic diagram in the lower right-hand corners of the lower panels indicates that the injection was carried out in both blastomeres at the two-cell stage. The insets inβ -catenin and β-catenin co-injection with Hex show Xnr3 staining in the animal hemisphere. Arrowheads indicate the site of injection. (F) Expression of β-catenin targets Siamois and Xnr3 in embryos depleted of endogenous Hex. Embryos were injected with a total of 40 ng Hex MO or control MO at the two-cell stage, either alone or in combination with 500 pg β-catenin. 500 pg mouse Hex (mHex) was used to rescue the phenotypes. Siamois (upper panel) and Xnr3 (lower panel) expression was analysed by in situ hybridisation at stage 10.5. Arrowheads indicate the ectopic expression induced by β-catenin.
Fig. 2. Downstream response to the induction of early embryonic signalling by Hex. (A) Real-time RT-PCR of Cerberus expression in VMZ explants analysed at stage 10.5. Embryos were injected with the indicated RNA and Cerberus expression quantified as in Fig. 1C. Data are based on three independent experiments. (B) In situ hybridisation for Cerberus expression and double in situ for Cerberus (purple) and nucGFP (light blue) expression at stage 10.5. Embryos were injected with 500 pg Hex, β-catenin or both at the two-cell stage into both blastomeres (indicated with a schematic diagram in the lower right-hand corner) or into a single-ventralblastomere at the four-cell stage (indicated by `V') alongside a nucGFP RNA. Arrowheads indicate the site of injection. (C,D) Real-time RT-PCR analysis of Goosecoid and Chordin. Embryos were injected as in A and RNA from either animal caps (C) or VMZ (D) explants extracted and analysed at stage 10.5. Values are normalised to the expression level of Odc and the relative change in gene expression for the genes analysed was calculated by dividing the values from injected samples by the values from the uninjected. Data is based on three independent experiments. (E) In situ hybridisation for Goosecoid and Chordin expression at stage 10.5. Embryos were injected with 500 pg Hex, β-catenin, or both, at the four-cell stage into a single-ventralblastomere alongside nucGFP. Double staining was performed. Arrowheads indicate the injected cells. High-magnification views of embryos co-injected with Hex and β-catenin are shown for both Goosecoid (indicated as Gsc) and Chordin (indicated as Chd) expression. (F) Depletion of Hex by Hex MO. Embryos were injected as in Fig. 1F and in situ hybridisation for Cerberus, Goosecoid and Chordin performed at stage 10.5. Arrowheads indicate the ectopic domain induced byβ -catenin injection.
Fig. 5. Validation of Hex targets in Xenopus embryos. (A) Expression of candidate Hex target genes in response to depleting endogenous Hex. In situ hybridisation for Xtle4, Xnr1, Xnr2, Xnr5 and Xnr6 in Hex MO depleted and rescued embryos. Embryos were injected as in Fig. 1F with the indicated MO/RNA combination and analysed by in situ hybridisation at stage 10.5. Where CHX is indicated, embryos were transferred to media containing CHX before MBT and collected 90 minutes later for fixation. As CHX slightly delays development, some of these embryos do not present evident dorsal lips. (B) Expression of candidate Hex target genes in response to Hex-λVP2. In situ hybridisation of embryos injected with Hex-λVP2 and assayed for the expression of the same set of candidate genes as in A. Embryos were injected at the four-cell stage on the dorsal side alongside nucGFP RNA. Embryos were bisected for better observation of the internal expression. Dorsal is towards the right.
Fig. 6. Xtle4 overexpression antagonises Hex-amplification of Wnt signalling. (A)In situ hybridisation for the expression of Xnr3 and Cerberus at stage 10.5. Embryos were injected at the two-cell stage in both blastomeres with 500 pg Hex, β-catenin and/or 3000 pg Xtle4. (B) Activity of Hex and β-catenin from the TOPflash Wnt reporter gene in the presence of Xtle4. The indicated DNA was co-transfected with either TOPflash or FOPflash in HEK 293T cells. Hex, Hex-λVP2 and β-catenin were used at 100 ng, Xtle4 at 90 ng. Values are represented as fold induction of the indicated reporters. Experiments were carried out in triplicate.
Fig. 7. Hex suppresses the activity of Nodal-related TGFβ proteins. (A) Axis duplication phenotypes induced by Hex and Xnr1. Embryos were injected into a single ventral-blastomere at the four-cell stage with 500 pg Hex, 250 pg Xnr1 or both and in situ hybridisation carried out at stage 35 using BF1. (B) Phenotypes induced by Hex and Xnr1 in ventral marginal zone explants. Embryos were injected with the indicated RNA at the two-cell stage, explants dissected at stage 10.5 and in situ hybridisation for BF1 carried out when staging control embryos reached stage 35. Hex was injected at 500 pg. Arrowheads indicate patches of BF1 expression. (C) In situ hybridisation for the expression of Cerberus at stage 10.5. Embryos were injected at the two-cell stage in both blastomeres with 500 pg Hex and/or 250 pg Xnr1 mRNA. (D) In situ hybridisation of Hex-depleted embryos for the mesendodermal markers Cerberus, Goosecoid and Chordin. Embryos were injected as in Fig. 1F. (E) Hex suppresses the induction of mesoderm in animal cap explants. Embryos were injected as in B with 500 pg Hex. Animal caps were dissected at blastula stage and cultured in 8 U/ml Activin protein until control embryos reached stage 18. (F) Molecular marker analysis in Hex-injected and Activin-treated animal cap explants. Animal caps were isolated from embryos injected as in B, cultured to stage 10.5 in 12 U/ml Activin, and RNA extracted for real-time RT-PCR analysis for Goosecoid, Chordin, Xbra and Mixer. Values are normalised to the expression level of Odc. Experiments were carried out in triplicate.
Fig. S1. Hex represses axial markers in Xenopus embryos. (A) In situ hybridisation of Hex-depleted embryos at stage 10.5 for Xlim1 (upper panel) and Xenopus Brachyury (Xbra) (lower panel). Embryos were injected with a total of 40 ng Hex MO or control MO at the two-cell stage. 500 pg mouse Hex were used to rescue the phenotypes. (B) In situ hybridisation for derriere at stage 10.5. Embryos were injected at the two-cell stage in both blastomeres with 500 pg Hex and/or 500 pg β-catenin.
tle4 (transducin-like enhancer of split 4)
gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage10.5, blastoporal view, dorsal up.
nodal5 (nodal homolog 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5, dorsal up.
nodal5 (nodal homolog 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, bissected NF stage 10.5, dorsal up.
nodal1 (nodal homolog 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5, dorsal up.
nodal1 (nodal homolog 1) gene expression in bissected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5, dorsal right.
nodal2 (nodal homolog 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5, dorsal up.
nodal2 (nodal homolog 2) gene expression in bissected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5, dorsal right.
nodal6 (nodal homolog 6) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5, dorsal right.
nodal6 (nodal homolog 6) gene expression in bissected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5, dorsal right.
tle4 (transducin-like enhancer of split 4) gene expression in bissected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5, dorsal right.
chrd.1 (chordin, gene 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5, dorsal up.
