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Experiment details for tbxt

Eimon PM and Harland RM (2002) Assay

Effects of heterodimerization and proteolytic processing on Derrière and Nodal activity: implications for mesoderm induction in Xenopus.

Gene Clone Species Stages Anatomy
tbxt.S laevis NF stage 10 to NF stage 10.25 mesoderm , marginal zone
tbxt.S laevis NF stage 11 mesoderm , marginal zone

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  Fig. 1. An analysis of the temporal and spatial expression pattern of derrière transcripts relative to other early markers of mesoderm and endoderm. (A) Analysis of temporal expression patterns by RT-PCR in Xenopus embryos. derrière transcripts are first detected at stage 8.5 at the same time as xnr1 and xnr4 as well as the homeobox gene mix1. Transcription of the mesodermal marker xbra and the dorsal specific marker cerberus are detected soon afterwards. (B-S) Whole-mount in situ hybridization analysis of Xenopus embryos at stages 8.5, 10+ and 11. The upper panels in each row show a representative embryo bisected through the animal-vegetal axis and oriented with the dorsal side on the right. The lower panels show a vegetal view of whole embryos, again oriented with dorsal sides to the right. (B-G) derrière, sox17β and bix4 transcripts are detected in distinct but overlapping domains at stage 8.5. Localized expression of xnr2, xbra and gsc is not apparent at this stage. (H-M) All transcripts show strong localized expression by stage 10+. derrière expression mirrors that of xbra, while xnr2 transcripts are restricted to the superficial cells of the marginal zone and greatly enriched on the dorsal side. (N-S) Expression in stage 11 embryos. Again derrière transcripts are detected throughout the region of the embryo expressing xbra. Arrowheads indicate the location of the dorsal blastopore lip in stage 10+ embryos.

Gene Clone Species Stages Anatomy
tbxt.S laevis NF stage 11 mesoderm , marginal zone

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  Fig. 2. derrière induces both dorsal and ventral markers of mesoderm in whole embryos. Embryos were injected with 200 pg of derrière (der) mRNA into a single blastomere at the four-cell stage. Injections were targeted to either the dorsal (dor) or ventral (vent) marginal zone and gastrula stage embryos were analyzed by whole-mount in situ hybridization. (A-F) Embryos viewed from the side of injection. (A-C) Expression of the mesodermal marker xbra is induced on the side of injection. (D-F) Dorsal injection leads to expanded gsc expression, while ventral injection causes no ectopic expression of gsc in the ventral marginal zone. (G-I) Embryos viewed from the vegetal pole with dorsal facing rightwards. Ventral injection of derrière mRNA leads to increased expression of the ventral and lateral marker xvent1, while dorsal injection has no effect.

Gene Clone Species Stages Anatomy
tbxt.S laevis NF stage 11 mesoderm , marginal zone

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  Fig. 3. A dominant negative derrière cleavage mutant (CM-der) inhibits mesoderm induction in Xenopus embryos. The effect of CM-der on mesoderm formation is similar to that of the short form of cerberus (cer-S) but differs markedly from the activity of the xnr2 cleavage mutant (CM-xnr2). Embryos were injected with either 2 ng of CM-der, 2 ng of CM-xnr2, or 500 pg of cer-S mRNA into a single blastomere at the four-cell stage along with 200 pg of a β-galactosidase lineage tracer. Stage 11 embryos were stained with red-gal to mark the site of injection and analyzed by whole-mount in situ hybridization. (A-D) Expression of the mesodermal marker xbra is inhibited in a similar manner by CM-der and cer-S; CM-xnr2 causes expansion of xbra into the animal hemisphere. (E-G) Expression of derrière is significantly diminished by cer-S but is expended into the animal hemisphere by CM-xnr2. (H-J) Xnr2 expression is strongly inhibited by cer-S and appears to be partially attenuated in the presence of CM-der. In H-J, embryos have been oriented with the site of mRNA injection at the top. (K) Mechanism of inhibition by cleavage mutant constructs. (L) derrière activity is blocked by both CM-der and cer-S in animal caps. Animal poles were injected at the one-cell stage with 200 pg derrière mRNA and co-injected with either 2 ng CM-der or 500 pg cer-S. By itself, derrière induces xbra and xnr1, as well as upregulating its own transcription. CM-der shows no mesoderm-inducing activity on its own and significantly reduces mesoderm induction by wild-type derrière. cer-S also blocks mesoderm formation in animal caps expressing derrière.

Gene Clone Species Stages Anatomy
tbxt.S laevis NF stage 11 mesoderm , marginal zone

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  Fig. 4. Cleavage mutant forms of Xnr2 retain diminished signaling activity and therefore do not function as authentic dominant negative molecules. (A) Diagram of the various Xnr2 cleavage mutant constructs tested. The gray region of the bar indicates the Xnr2 proA and proB regions, while the white regions represent the mature Xnr2 ligand. The Activin pro domain is indicated in blue. (B) RT-PCR analysis of xnr2 cleavage mutant constructs. Embryos were injected in the animal pole at the one-cell stage with 10 pg of xnr2 or 2 ng of the cleavage mutant mRNAs. Wild-type xnr2 and both CM-xnr2 and DCM-xnr2 induce mesoderm in animal caps, as indicated by the presence of xbra transcripts. An equivalent dose of CM-der shows no mesoderm inducing activity. Both xnr2 cleavage mutant constructs fail to induce extreme dorsal fates (marked by gsc). (C-T) xnr2 cleavage mutant constructs induce ectopic mesoderm in whole embryos. Embryos were injected with the 10 pg xnr2 or 2 ng cleavage mutant mRNAs in the animal pole at the one-cell stage, allowed to develop to stage 11 and analyzed by in situ hybridization. Wild-type xnr2 and all three cleavage mutant constructs cause expansion of the mesodermal marker xbra, the dorsal mesodermal marker gsc and the ventral/lateral mesodermal marker xwnt8 (F-Q). By contrast, CM-der leads to an inhibition of xbra expression and has no obvious effect on gsc and xwnt8 (R-T), presumably because of limited diffusion of the animally injected mRNA).

Gene Clone Species Stages Anatomy
tbxt.S laevis NF stage 11 mesoderm , marginal zone

  Fig. 6. Cleavage mutant forms of Xnr2 are secreted and can act non-cell autonomously. (A-D) DCM-Xnr2 induces mesoderm non-cell autonomously. Four-cell embryos were injected at the animal pole with (A) 100 pg xnr2 + 200 pg β-galactosidase, (B) 2 ng DCM-xnr2 + β-galactosidase or (C) lacZ/adr2 mRNA. lacZ expression was visualized by red-gal staining and xbra expression was detected by in situ hybridization. (D) Control embryos. (E) CM-Xnr2 and DCM-Xnr2 are secreted from oocytes while CM-Der is not. Mature oocytes were injected with 25 ng of the indicated mRNAs and labeled with [35S]methionine. CM-Xnr2 and DCM-Xnr2 are efficiently secreted while CM-Der is retained within the oocyte lysate (asterisk). Both Xnr2 and CM-Xnr2 undergo proteolytic processing, as indicated by the presence of the proB domain (arrowhead). Mutation of both cryptic and canonical sites in DCM-Xnr2 abolishes all proteolytic processing.