April 1, 2010;
T-box family transcription factors play many roles in Metazoan development. Here we characterise Tbx6r
, a unique Tbx6
paralogue isolated from the amphibian Xenopus. The evolution and developmental integration of this divergent T-box gene within the vertebrates reveals an unexpected level of plasticity within this conserved family of developmental regulators. We show that despite their co-expression, Tbx6
have dissimilar transcriptional responses to ligand treatment, and their ability to activate ligand expression is also very different. The two paralogues have distinct inductive properties: Tbx6
neural markers. We use hybrid proteins in an effort to understand this difference, and implicate the C-terminal regions of the proteins in their inductive specificities. Through loss-of-function analyses using antisense morpholino oligonucleotides we show that both Tbx6
paralogues perform essential functions in the development of the paraxial and intermediate mesoderm
and the neural crest
in Xenopus. We demonstrate that Tbx6
both induce FGF8
expression as well as that of pre-placodal markers, and that Tbx6
can also induce neural crest
markers via a ligand-dependent mechanism involving FGF8
. Our data thus identify an important new function for this key developmental regulator.
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Fig. 5. Tbx6r depletion using antisense morpholino oligonucleotides causes developmental defects in X. laevis. (A) Diagram illustrating positions of the various morpholinos used relative to the Tbx6r mRNA. The translational initiation site is underlined and the T-domain is highlighted in red. MO3 is an exact match to the RNA sequence because it was designed as a translation blocking morpholino although it acts as a splice-blocker, whereas MO4 is an inexact match because the 3′ region targets intronic sequence. (B) RT-PCR analysis of splicing morpholino efficacy and specificity in stage 19 embryos injected with 50 ng morpholino. Gene-specific primers span the indicated introns (Int). Asterisks indicate unspliced products. pTbx6 and pTbx6R are positive control PCR products generated from plasmid DNA. (C–E) Embryos injected with 90 ng of indicated morpholino. (F, G) Embryos injected with 30 ng morpholino.
Fig. 6. Phenotypic defects resulting from depletion of Tbx6r and Tbx6 in X. tropicalis. (A) Efficacy of splicing morpholino in siblings of embryos pictured in B–E. The upper band is of a size consistent with an unspliced product and may represent pre-mRNA since it is present in all samples. XtTbx6r MO inhibits splicing significantly but incompletely. Tbx6r splicing is not affected in Tbx6 morphants. Embryos were injected at the one-cell stage with the following morpholinos: 30 ng GeneTools control (B), 15 ng XtTbx6r (C), 30 ng XtTbx6r (D), 30 ng XtTbx6 (E). Tail lengths were measured from the proctodeal opening to the tail tip at stage 41. Mean values ± the standard error of the mean are shown in (F). Measurements were subjected to ANOVA followed by Scheffe's Test of Least Significant Difference. The annotations a, b and c above the bars on the graphs represent statistically significant differences between groups at p < 0.05.
Fig. 7. Mesoderm patterning in Tbx6r-depleted embryos. Embryos were injected unilaterally at the 2-cell stage with 25 ng of the indicated morpholino. The probes used are indicated on the images. All images are of in situ hybridisations except J and K, which are antibody stains. Asterisks indicate injected sides. (A–F) vegetal views of gastrulae; (G–I) dorsal views of neurula; (J–K) dorsal views of tailbud embryos, anterior to left. (L–O) are lateral views of hemi-injected tailbud embryos.
Fig. 8. Neural patterning in Tbx6r-depleted embryos. All embryos were unilaterally injected at the 2-cell stage with 25 ng indicated morpholino except A–C, which were injected with 50 ng at the 1-cell stage. The morpholinos were lissamine-labelled to allow determination of injected side marked by an asterisk (M–O are fluorescent images of the embryos in J–L). In situ probes used are indicated on the figures. (A–C) dorsal views of neurula, anterior at bottom; (D–R) anterior views of neurula; (S–V) lateral views of tailbud embryos; T, V are injected sides of embryos in S, U respectively.
Fig. 9. Embryos lacking Tbx6 have defects in neural crest and paraxial mesoderm formation. (A) Test of Tbx6 splicing morpholino (T6sp1) efficacy and specificity by RT-PCR. The 226 bp amplicon indicating correct splicing of Tbx6 is greatly reduced in stage 16 embryos injected with 50 ng T6sp1 MO but unaffected in siblings injected with 50 ng Tbx6r MO4. (B–E) Morphant phenotypes of Tbx6 translation-blocker (T6 ATG MO) and splice-blocker (T6sp1 MO) morpholinos. (F–R) In situ analysis of embryos unilaterally injected (*) with 25 ng T6sp1 MO at the 2-cell stage. F, J, L, N were injected with control morpholino; G, K, M, O were injected with T6sp1 MO. H and I are fluorescent images of the anterior views of the neurula depicted in F and G respectively, showing distribution of the lissamine-labelled morpholino. P and Q show uninjected and T6sp1 MO-injected sides of the same tailbud embryo; R and S are the equivalent images of a control-injected morphant. J, K: dorsal views; L–O anterior views.
Fig. 10. Tbx6 and Tbx6r induce FGF8 and neural plate border markers. (A) Tbx6 expression and (B) Tbx6r expression in the mesoderm of parasagittally bisected stage 15 embryos co-stained with the neurectodermal marker Krox20. (C) Differential ligand-inducing activities of Tbx6, Tbx6r and the hybrid constructs in gastrula-stage caps. 500 pg of each RNA was injected. (D) FGF8 is induced by 500 pg Tbx6r–MT in stage 23 caps. (E) Neural plate border induction in stage 23 animal caps. (F) Inhibition of Tbx6-mediated neural plate border induction by 500 pg Tbx6–MT RNA in stage 23 animal caps by 50 ng morpholino targeting either FGF8 or Wnt8. Experiments were independently duplicated (C, D, F) or triplicated (E); representative results of individual experiments are shown. Control caps in (C) to (F) were derived from uninjected embryos.
Supplementary Fig. 1. Comparative spatiotemporal expression analysis of Tbx6r, Tbx6 and VegT/apod. (A) Temporal analysis of the expression of the three genes during X. laevis development measured by quantitative RT-PCR. (B-P) Spatial analysis of gene expression by in situ hybridisation. B, G, L: side view; C, H, M: vegetal view; D, E, I, J, N, O: dorsal view, anterior to left; F, K, P: lateral view, anterior to left.
Supplementary Fig. 2. The biological activity of Tbx6r is atypical of the Tbx6 sub-family. (A) Determination of translational start codon by western analysis. Lanes contain lysates from early gastrula stage 10 embryos injected at the 1-cell stage with 500 pg RNA made from the wild-type myc-tagged Tbx6r-MT construct or from derivatives in which either of the first two methionines were mutated (M1R and M20R). All injected samples were spiked with 500 pg Xbra-HA as translation control and the blot was probed sequentially with anti-myc, anti-HA and anti-GAPDH antibodies, the last of these acting as a loading control. (B) Expression of anterior neural markers in animal caps cultured to stage 23 with the RNA amount injected into the 1-cell embryo specified in picograms. Expression in all samples was normalised to that of ornithine decarboxylase (ODC) as was the case for all subsequent experiments. The animal cap assay was performed three times and results of a representative experiment are shown. Here, the level of induction depicted is relative to that caused by 400 pg noggin RNA. (C) Comparison of translation efficiency of the Tbx6r-MT and Tbx6-MT constructs in NF10 embryos; siblings to those shown in D. (D) Expression of mesodermal and endodermal markers in NF 11.5 animal caps. Induction is depicted relative to that caused by 250 pg Tbx6. Control caps in (B) and (D) were derived from uninjected embryos.
Isolation and characterization of two T-box genes from sponges, the phylogenetically oldest metazoan taxon.