February 15, 2006;
FGF8, Wnt8 and Myf5 are target genes of Tbx6 during anteroposterior specification in Xenopus embryo.
The T-box transcription factor Tbx6
is required for somite
formation and loss-of-function or reduced activity of Tbx6
result in absence of posterior paraxial mesoderm
or disorganized somites
, but how it is involved in a regulatory hierarchy during Xenopus early development is not clear. We show here that Tbx6
is expressed in the lateral
and ventral mesoderm
of early gastrula
, and it is necessary and sufficient to directly and indirectly regulate the expression of a subset of early mesodermal and endodermal genes. Ectopic expression of Tbx6
inhibits early neuroectodermal gene expression by strongly inducing the expression of posterior
mesodermal genes, and expands the mesoderm
territory at the expense of neuroectoderm
. Conversely, overexpression of a dominant negative Tbx6
mutant in the ventral mesoderm
inhibits the expression of several mesodermal genes and results in neural induction in a dose-dependent manner. Using a hormone-inducible form of Tbx6
, we have identified FGF8
as immediate early responsive genes of Tbx6
, and the induction of these genes by Tbx6
is independent of Xbra
. These target genes act downstream and mediate the function of Tbx6
in anteroposterior specification. Our results therefore identify a regulatory cascade governed by Tbx6
in the specification of posterior mesoderm
during Xenopus early development.
[+] show captions
Fig. 1. Overlapping expression of Tbx6, XMyf5, XMyoD and Xwnt8 at the early gastrula stage analyzed by in situ hybridization. (A) Tbx6 is expressed in the lateral and ventral mesoderm of early gastrula. (B) XMyf5 shows similar expression pattern but is expressed at a lower level in the most ventral region. (C) XMyoD is expressed in a very similar pattern as Tbx6. (D) The expression of Xwnt8 is localized to the ventral region with a relatively lower level in the lateral mesoderm. The expression of all four genes is excluded in the dorsal region.
Fig. 2. Regulation of mesodermal and neural gene expression by Tbx6 in marginal zone explants. (A) Embryos at 4-cell stage were injected with Tbx6VP16 mRNA in the DMZ region or with Tbx6EnR mRNA in the VMZ region. Injected embryos were cultured to early gastrula stage for dissection of DMZ and VMZ explants. (B) Gene expression in DMZ and VMZ explants at the early gastrula stage. Tbx6 inhibits the expression of dorsal genes like Otx2 and induces the expression of ventral genes like Xwnt8 and szl in the DMZ explants. In contrast, Tbx6EnR induces the expression of dorsal genes like Otx2 in the VMZ explants, whereas it inhibits the expression of latero-ventral genes including Xbra, szl, Xwnt8, XMyoD and FGF8. (C) Gene expression in DMZ and VMZ explants expressing Tbx6VP16 or Tbx6EnR at early tail-bud stage. Tbx6 inhibits the expression of Otx2 and N-CAM, but up-regulates posterior genes Hoxb9, Xcad2 and Mespo in DMZ explants. Tbx6EnR induces the expression of neural markers XAG1, Otx2, En2 and N-CAM in VMZ explants, while it inhibits the expression of posterior genes including Hoxb9, Xcad2 and Mespo.
Fig. 3. Dose-dependent effect of Tbx6EnR on gene expression. Following injection of different amounts of Tbx6EnR mRNA in the ventral region at 4-cell stage, VMZ explants were dissected and RT-PCR was performed at stage 10.5 and 25. The intensity of each PCR product was normalized to ODC. This experiment was performed in triplicates with similar result.
Fig. 4. Expression of mesoderm, endoderm and neural markers in Tbx6VP16-injected early gastrula. Embryos at 4-cell stage were dorsally injected with Tbx6VP16 mRNA and cultured to early gastrula stage for in situ hybridization. (A) Vegetal view of an uninjected early gastrula. (B) Dorso-vegetal view of a Tbx6VP16-injected early gastrula with a condensation of pigmentation near injection sites. (C) Expression of Xbra in an uninjected early gastrula. (D) A Tbx6VP16-injected early gastrula showing a shift of Xbra expression domain to more anterior region. Arrowheads indicate the blastopore. (E) XMyf5 expression in an uninjected early gastrula. (F) Ectopic expression of XMyf5 in the dorsal region induced by Tbx6VP16. (G) Xwnt8 expression in an uninjected early gastrula. (H) Ectopic expression of Xwnt8 in a Tbx6VP16-injected early gastrula. (I) FGF8 expression in an uninjected early gastrula is localized to the entire marginal zone. (J) Dorsal injection of Tbx6VP16 shifted FGF8 expression to a more anterior region. (K) Sox17α expression is located in the yolk plug and latero-ventral blastoporal lip with lower level in the dorsal region. (L) Tbx6VP16 strongly induces ectopic Sox17α expression in the dorsal region. (M) Expression of the neural marker Sox3 in the dorsal ectoderm of an uninjected early gastrula. (N) Absence of Sox3 expression in the dorsal ectoderm following injection of Tbx6VP16.
Fig. 5. Mesoderm and endoderm induction in ectoderm explants by Tbx6. Embryos at 4-cell stage were injected with Tbx6VP16 or Tbx6 mRNA in the animal pole region and ectodermal explants were cultured to stage 14. (A) Control uninjected explants. (B) Tbx6VP16-injected explants show elongation and protrusions. (C) Section from a Tbx6VP16-injected explant, low magnification. (D) Section from a Tbx6VP16-injected explant, high magnification. (E) RT-PCR analysis of mesoderm and endoderm markers. Both Tbx6VP16 and Tbx6 induce the expression of endodermal marker Sox17α in addition to latero-ventral mesoderm markers.
Fig. 6. Tbx6 inhibits neural induction in vitro and in vivo. (A) Tbx6 counterbalances the neuralizing activity of chordin. Embryos were injected with indicated mRNA and ectodermal explants were cultured to stage 25 for RT-PCR analysis. Overexpression of chordin induces the expression of anterior neuroectoderm markers Otx2 and XAG1, as well as the pan-neural marker N-CAM. Coexpression of Tbx6VP16 with chordin strongly inhibits neural induction and induces the expression of different posterior mesoderm markers including Hoxb9, Xcad2 and Mespo, in addition to somitic mesoderm markers muscle actin and XMyf5. LacZ mRNA was either injected alone or coinjected with Tbx6VP16 or with Tbx6EnR mRNA in the indicated region at 8-cell stage. (B) Distribution of βgal-stained cells in the head and ventral regions in embryos injected with LacZ mRNA alone in the animal pole region of the two dorso-animal blastomeres. (C) Distribution of βgal-stained cells in the trunk region and somitic mesoderm following coinjection of LacZ mRNA with Tbx6VP16 mRNA in the animal pole region of the two dorso-animal blastomeres. (D) Section at the head region of an embryo in panel B. (E) Section at the trunk region from an embryo in panel C. (F) βgal-stained cells are populated to the head region and somitic mesoderm when LacZ mRNA is injected alone in the marginal zone of the two dorso-animal blastomeres. (G) Coinjection of Tbx6EnR mRNA with LacZ mRNA in the marginal zone of the two dorso-animal blastomeres prevents somitic distribution of βgal-stained cells. (H) Section at the trunk region from an embryo in panel F. (I) Section at the head region from an embryo in panel G.
Fig. 7. Identification of Tbx6 direct targets genes. (A) Schematic representation of the inducible construct. (B) Embryos at 4-cell stage were injected with Tbx6VP16-GR mRNA and ectodermal explants were dissected and cultured to stage 10.5. Both uninjected and injected explants were treated with cycloheximide (CHX) or dexamethasone (DEX), or both, for 1.5 h. The expression of a panel of mesoderm and endoderm markers was analyzed by RT-PCR. Among these genes, the expression of FGF8, Xwnt8 and XMyf5 was rapidly and specifically induced and was not blocked in the presence of CHX. (C) Tbx6-induced expression of XMyf5, Xwnt8 and FGF8 was blocked by Tbx6EnR in a dose-dependent manner, but not by XbraEnR and VegTEnR. (D) Tbx6EnR strongly blocks the expression of XMyf5, Xwnt8 and FGF8 induced by Xbra and VegT. XbraEnR and VegTEnR also block the activity of Xbra and VegT, respectively.
Fig. 8. Functional interaction between Tbx6, FGF8, Xwnt8 and XMyf5. (A–D) Interaction between Tbx6 and Xwnt8 in anteroposterior patterning. Embryos were injected at 4-cell stage and cultured to stage 35. (A) A control embryo. (B) dorsal(C) A Xwnt8Mo-injected embryo in the ventral region with enlarged head and shortened anteroposterior axis. (D) A Tbx6VP16-GR-injected embryo incubated in DEX at stage 11 shows anterior deficiency at stage 35. (E) Coinjection of Xwnt8Mo with Tbx6VP16-GR mRNA rescues head development. (F) Interaction between Tbx6, Xwnt8 and XMyf5 in myogenesis. Embryos were injected at 2-cell stage and animal cap explants were cultured to stage 11 for RT-PCR analysis. Inhibition of Xwnt8 and XMyf5 activity strongly inhibits XMyoD expression induced by Tbx6VP16. (G) FGF8 exhibits similar activity as Tbx6VP16 in inhibiting neural induction in explants cultured to stage 25. A dominant negative FGF receptor (XFD) inhibits XMyf5 and Xcad2 expression induced by Tbx6VP16 in explants cultured to stage 25.
Table 1. Functional interaction between Tbx6 and Xwnt8 in anteroposterior patterning. Embryos were injected at 4-cell stage and cultured to stage 35 for score of phenotypes. Acephalic embryos show absence of cement gland and eyes, microcephalic embryos exhibit reduced head structures with smaller cement gland and eyes, and embryos with enlarged head display strongly dorsalized phenotype with enlarged cement gland and reduced trunk and posterior regions. The results were expressed as percentage, except n, which refers to total embryos scored from three independent experiments.
Fig. 9. Regulatory hierarchy in posterior mesoderm specification and myogenesis involving Tbx6, FGF8, Xwnt8 and XMyf5. Tbx6 functions upstream of these genes in both processes (see Discussion for detail).