QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by White, R. J. Articles by Smith, J. C. Search for Related Content PubMed PubMed Citation Articles by White, R. J. Articles by Smith, J. C.

Direct and indirect regulation of derrière, a Xenopus mesoderm-inducing factor, by VegT

R. J. White¹, B. I. Sun^2,*, H. L. Sive² and J. C. Smith^1,

¹ Division of Developmental Biology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK and Wellcome Trust/Cancer Research UK Institute and Department of Zoology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
² Whitehead Institute and Massachusetts Institute of Technology, Nine Cambridge Center, Cambridge, MA 02142-1479, USA
^* Present address: Merck & Co. Inc., PO Box 4, West Point, PA 19486, USA

View larger version (78K): [in a new window]   Fig. 1. Comparison of the expression patterns of derrière (A-C) and VegT (D-F) at stages 9 (A,D), 10.5 (B,E) and 12 (C,F). Embryos were fixed at the indicated stages and processed for in situ hybridisation. Note the similarities between the expression patterns of the two genes.

View larger version (49K): [in a new window]   Fig. 2. Derrière, like activin, but unlike Xnr-2, can exert long-range effects in Xenopus tissue. (A) The experimental design. Animal pole regions dissected from embryos uniformly labelled with the cell lineage marker fluorescein-lysine-dextran (FLDx) were juxtaposed with animal pole regions derived from embryos injected with RNA encoding activin (5 pg), derrière (500 pg), or Xnr-2 (500 pg). Caps were cultured for 3 hours and then examined by whole-mount in situ hybridisation for expression of Xbra (B,D,F,H) or by fluorescence microscopy for detection of FLDx (C,E,G,I). (B,C) A control conjugate in which an FLDx-labelled animal cap was juxtaposed with an uninjected cap. Xbra is not activated. (D,E) A conjugate in which an FLDx-labelled animal cap was juxtaposed with an cap derived from an embryo injected with RNA encoding activin. Note induction of Xbra in FLDx-labelled tissue, as previously described (Gurdon et al., 1994; Jones et al., 1996). (F,G) A conjugate in which an FLDx-labelled animal cap was juxtaposed with an cap derived from an embryo injected with RNA encoding Xnr-2. Induction of Xbra is restricted to unlabelled tissue (see Jones et al., 1996). (H,I) A conjugate in which an FLDx-labelled animal cap was juxtaposed with a cap derived from an embryo injected with RNA encoding derrière. Note induction of Xbra in both unlabelled and FLDx-labelled tissue. In this respect the pattern of Xbra expression differs from that induced by activin (see text).

View larger version (55K): [in a new window]   Fig. 3. Derrière is a direct target of VegT. VegT-GR, a hormone-inducible form of VegT, was injected into Xenopus embryos at the one-cell stage and the embryos were allowed to develop to the late blastula stage. Animal caps were dissected from these embryos and they were incubated in dexamethasone (DEX), cycloheximide (CHX) or both. After 3 hours of culture they were analysed for expression of derrière or Bix4 by RNAase protection. Ornithine decarboxylase (ODC) was used as a loading control. Note that both derrière and Bix4 are induced by DEX in the presence of CHX, but that CHX causes a decrease in levels of activation of derrière.

View larger version (22K): [in a new window]   Fig. 4. T-box and Fast binding sites in the derrière promoter. (A) Schematic diagram of the derrière genomic fragment obtained in this work. It comprises 851 base pairs 5' of the first exon, the whole of exon 1 (247 base pairs) and 844 base pairs of the first intron. F1 and F2 represent the putative Fast sites and Tbs1 and Tbs2 the putative T box sites. (B) Partial sequence of the derrière promoter region. The transcription start site is indicated by an arrow and the beginning of the protein coding region is shown in lower case. T box sites Tbs1 and Tbs2 are underlined, as are two matches to the consensus Fast binding site (F1 and F2). (C) Comparison of Tbs1 and Tbs2 with the T box binding site deduced from binding site selection experiments (Kispert and Herrmann, 1993; Conlon et al., 2001).

View larger version (42K): [in a new window]   Fig. 5. The derrière 5' regulatory region drives mesendodermal expression of a GFP reporter gene. (A) The reporter construct. (B-D) Views of transgenic Xenopus embryos showing expression of GFP by in situ hybridisation. (B) Stage 10.5 embryo, vegetal view. Expression of GFP is detectable in the mesoderm and (weakly) in the endoderm. (C) Dorsal view of the embryo shown in B. (D) Lateral view of a stage 10.5 embryo with dorsal blastopore lip to the right. This embryo was bisected before the staining procedure. Note that expression of the reporter construct differs from that of the endogenous gene in that transcripts persist in the involuted mesoderm.

View larger version (68K): [in a new window]   Fig. 6. Electrophoretic mobility shift assay demonstrating that VegT binds Tbs1. A 32P-labelled 36 base pair probe including Tbs1 was incubated with uncharged reticulocyte lysate (lanes 1-3) or with in vitro translated HA-tagged VegT (lanes 4-9). Assays in lanes 2, 5 and 8 included an excess of unlabelled probe as competitor (+) and assays in lanes 3, 6 and 9 included an excess of unlabelled mutated probe (M). Supershift assays in lanes 7-9 included a rat anti-HA monoclonal antibody, with incubation carried out for 10 minutes at 4°C. A specific shift is visible in lanes 4 and 6, and these are ‘supershifted’ in lanes 7 and 9.

View larger version (40K): [in a new window]   Fig. 7. Mutational analysis of the putative T-box binding sites in the derrière promoter. (A) Diagrams showing the constructs used and the mutations made in the T-box binding sites. (B) Luciferase assay in animal caps showing that induction of the firefly luciferase reporter gene is independent of the T-box sites. (C-F) Vegetal views of stage 10.5 transgenic Xenopus embryos expressing the indicated transgenes. Note that all four constructs are expressed in the mesendoderm. Expression of the Tbs1,2 construct in the case illustrated in F is higher in the endoderm than the mesoderm, but this is not a consistent observation.

View larger version (77K): [in a new window]   Fig. 8. Electrophoretic mobility shift assay demonstrating that Fast-1 binds the Fast sites identified in Fig. 4. A 32P-labelled 36 base pair probe including both Fast sites was incubated with uncharged reticulocyte lysate (lane 2) or with in vitro translated Flag-tagged Xfast-1 (lanes 4-7). The assay in lane 4 included an excess of unlabelled probe as competitor (WT) and the assay in lane 5 included an excess of unlabelled mutated probe (Mut). Supershift assays included an anti XFast-1 antibody (lane 6) and an anti Flag antibody (lane 7).

View larger version (15K): [in a new window]   Fig. 9. The derrière 5' regulatory region responds to activin. (A) Deletion analysis shows that an activin-responsive element lies between –441 and –139 nucleotides in a region that includes the two Fast sites. (B) Targeted mutation of the T box and Fast sites suggests that activin induction can also occur in an indirect fashion, through the T box sites.

View larger version (14K): [in a new window]   Fig. 10. (A) The initial phase of reporter gene activation in response to VegT requires intact T box sites. The indicated reporter constructs were injected into Xenopus eggs at the one-cell stage along with the VegT RNA or, as a control, lacZ RNA. Animal caps were dissected at stage 9 and cultured for 2 or 3 hours, as indicated. Analysis of luciferase activity shows that reporter gene activity was abolished by mutation of the T box sites in the 2-hour time-point, but not in the 3-hour time-point. (B) Activation of the indicated reporter constructs in Xenopus oocytes. Mutation of Tbs1 causes a dramatic reduction in luciferase activity and mutation of both sites abolishes the ability to respond to VegT.

View larger version (68K): [in a new window]   Fig. 11. Inhibition of activin-like or FGF signalling does not interfere with activation of derrière but does prevent its maintenance. Embryos were injected at the two-cell stage with 800 pg RNA encoding the truncated activin receptor XAR (A,D), the truncated FGF receptor XFD (B,E), or the control construct d50 (C,F). RNA encoding ß-galactosidase was co-injected as a cell lineage label in all cases. Embryos were allowed to develop to stage 9.5 (A-C) or 11 (D-F) and expression of derrière was analysed by in situ hybridisation. The initial activation of derrière is not affected by inhibition of activin-like (A) or FGF (B) signalling; note the overlap of ß-galactosidase staining (blue) with the in situ hybridisation reaction product (purple), which at this early stage is largely nuclear. Continued expression of derrière, however, does require activin-like and FGF signalling; note down-regulation of expression in D and E. Vg, vegetal pole.

View larger version (9K): [in a new window]   Fig. 12. A network of interactions in the regulation of derrière. Solid black arrows indicate direct actions of VegT and dotted black arrows indicate effects of VegT that may be indirect. Solid grey arrows indicate probable direct interactions of derrière and nodal-related gene products on the derrière promoter, which occur after the initial effects of VegT. Dotted grey arrows indicate the effects of gene X on the derrière promoter and of the nodal-related proteins on the VegT 5' regulatory region, causing the activation of Antipodean. Gene X is yet to be identified, but may represent a member of the FGF family. See text for details.