spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Eimon, P. M.
Right arrow Articles by Harland, R. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Eimon, P. M.
Right arrow Articles by Harland, R. M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

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

Peter M. Eimon and Richard M. Harland*

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202, USA



View larger version (68K):

[in a new window]
  		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, sox17ß 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.

 


View larger version (70K):

[in a new window]
  		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.

 


View larger version (55K):

[in a new window]
  		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 ß-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.

 


View larger version (68K):

[in a new window]
  		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).

 


View larger version (49K):

[in a new window]
  		Fig. 5. Loss of the cryptic proteolytic cleavage site in Xnr2 has no impact on activity. (A) RT-PCR analysis of the xnr2 upstream cleavage mutant construct (UCM-xnr2). Embryos were injected in the animal pole at the one-cell stage with 1 pg or 10 pg of xnr2 or UCM-xnr2 mRNA. Both molecules induce the mesoderm markers xbra and vegT at equivalent doses (lanes 1 and 4). (B-E) Analysis of UCM-xnr2 in whole embryos. Embryos were injected at the one-cell stage with 0.1 pg or 1 pg of xnr2 or UCM-xnr2 mRNA and allowed to develop to stage 20.

 


View larger version (69K):

[in a new window]
  		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 ß-galactosidase, (B) 2 ng DCM-xnr2 + ß-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.

 


View larger version (40K):

[in a new window]
  		Fig. 7. CM-Der can antagonize Nodal-mediated mesoderm induction. (A) In whole embryos, CM-der attenuates xnr2 overexpression phenotypes. One-cell embryos were injected at the animal pole with increasing doses of xnr2 mRNA (top row; xnr2 levels range from 1 to 10 pg). The overexpression phenotype is shown at stage 11 (left side of each panel) and stage 20 (right side of each panel). This overexpression phenotype is markedly decreased by co-injection of 3 ng of CM-der mRNA (bottom row). (B) In animal caps, CM-der antagonizes both xnr2-mediated mesoderm induction and the induction of other TGFß transcripts. Embryos were injected at the animal pole at the one-cell stage with increasing amounts of xnr2 mRNA (0.1, 1 and 10 pg) either alone or with 2 ng of CM-der mRNA. Co-injection of CM-der attenuates mesoderm induction by xnr2 (as indicated by xbra, gsc and chordin) and also retards the induction of multiple TGFß transcripts.

 


View larger version (47K):

[in a new window]
  		Fig. 8. CM-Der inhibits secretion of multiple members of the TGFß superfamily. Oocytes were injected with synthetic mRNA encoding epitope tagged proAct-Xnr2 (HA tagged), proAct-Derrière (Flag tagged) or BMP4 either alone or in combination with CM-Der. Injected oocytes were cultured in the presence of [35S]methionine and the supernatants and lysates analyzed by immunoprecipitation. Supernatants from oocytes expressing proAct-Xnr2, proAct-Derrière or BMP4 alone contain bands corresponding to the predicted sizes of the mature ligands, while the lysates also contain higher molecular weight unprocessed precursor proteins (lanes 1-3). CM-Der (tagged with the Glu-Glu epitope) is seen only in oocyte lysates and in its presence no mature Xnr2, Derrière or BMP4 ligands are detected in the supernatants (lanes 4-7). Unprocessed precursor proteins are still detected in oocyte lysates. Asterisks indicate unprocessed BMP4; arrowheads indicate mature Xnr2 and Derrière ligands.

 


View larger version (53K):

[in a new window]
  		Fig. 9. CM-der induces neural tissue in ectodermal explants. Embryos were injected at the animal pole at the one-cell stage with 25 pg noggin mRNA or 2 ng CM-der mRNA and explants were cultured until stage 20 and analyzed by RT-PCR. Both noggin- and CM-der-treated animal caps express the neural markers nrp-1 and n-cam in the absence of muscle actin (MA, a mesoderm-specific transcript) and at the expense of epidermal keratin (EK, an epidermal marker).

 


View larger version (48K):

[in a new window]
  		Fig. 10. Derrière physically associates with other members of the TGFß superfamily, including Xnr2 and BMP4. (A) HA-tagged proAct-Derrière was co-expressed in Xenopus oocytes along with either Flag-tagged proAct-Xnr2 or untagged proAct-Xnr2. When HA-tagged proAct-Derrière is expressed on its own and immunoprecipitated with {alpha}HA, the mature ligand is seen as a broad range band between 22 and 28 kDa (lane 3). In the presence of Flag-tagged proAct-Xnr2, two distinct bands are apparent (lane 4); the removal of the Flag epitope tag from proAct-Xnr2 (a decrease in molecular weight of approximately 1 kDa) causes a corresponding mobility shift in the upper of the two bands (mature Xnr2 bands marked by asterisks). Note that co-expression of Derrière and Xnr2 also causes an overall decrease in the molecular weight of the mature Derrière ligand. (B) Xnr2 and BMP4 co-precipitate with Derrière. HA-tagged proAct-Derrière was expressed in oocytes either alone or in combination with Flag-tagged proAct-Derrière, proAct-Xnr2 or BMP4. Supernatants were analyzed by two-step immunoprecipitation; proteins were initially pulled down with {alpha}HA coupled to protein A, released by boiling and immunoprecipitated a second time using {alpha}Flag. Flag-tagged Derrière, Xnr2 and BMP4 are detected after the double immunoprecipitation (lanes 5-7), indicating that all of them associate with HA-tagged Derrière. All proteins are efficiently translated and secreted into the supernatant, as demonstrated by single immunoprecipitations (top two panels). (C) Wild-type Xnr2 co-precipitates with wild-type Derrière in oocyte lysates. Flag-tagged Derrière was expressed either alone or in combination with HA-tagged Derrière or HA-tagged Xnr2. Lysates were analyzed by {alpha}HA and {alpha}Flag immunoprecipitations. In both cases, unprocessed Xnr2 and Derrière were found to associate (lane 5). In addition, low levels of the mature Flag-tagged Derrière ligand co-precipitated with HA-tagged Xnr2 (arrowhead, bottom {alpha}HA panel).

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2002