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A novel Xenopus Smad-interacting forkhead transcription factor (XFast-3) cooperates with XFast-1 in regulating gastrulation movements

Laboratory of Developmental Signalling, Cancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK

View larger version (60K): [in a new window]   Fig. 1. XFast-3 is a novel member of the Fast family. (A) ARF2 is an ARE-binding complex distinct from the XFast-1-containing ARF1, which appears about 240 minutes post stage 8. Nuclear extracts prepared from uninjected embryos or embryos injected with 200 pg mRNA encoding activin-ßA at the times indicated were analysed by bandshift using the ARE as probe. ARF1 is only detected in activin-injected embryos; the faster mobility complex ARF2 is detected in uninjected embryos. (B) The sequence of XFast-3 aligned with the sequences of XFast-1, mouse Fast-2/FoxH1, human Fast-1/FoxH1 and zebrafish FoxH1 (Attisano et al., 2001). Grey underlining, forkhead/winged-helix DNA-binding domain (Kaufmann and Knöchel, 1996); broken line, the Fast motif (FM); unbroken black line, the Smad interaction motif (SIM) (Germain et al., 2000).

View larger version (39K): [in a new window]   Fig. 2. XFast-3 is the transcription factor component of endogenous ARF2. (A) Whole cell extracts were prepared from NIH3T3 cells transfected with either Flag-tagged XFast-1 or XFast-3 that were either treated or not with TGFß1 as indicated. Extracts were analysed by bandshift assay on the ARE and complexes supershifted with monoclonal antibodies against Flag, Smad2/3 or Smad4. The Smad-containing complexes ARF1 and ARF2 are indicated, as are the supershifted complexes and the complex of XFast-1 with DNA. (B) NIH3T3 cells were transfected with ARE-luciferase, EF-lacZ as an internal control and plasmids expressing transcription factor (XFast-1 or XFast-3), as indicated. Cells were treated with TGFß1 for 8 hours, then harvested and luciferase activity was measured relative to ß-galactosidase activity from the internal control. The data are averaged from six independent experiments and standard deviations are shown. (C) Nuclear extracts were prepared from uninjected Xenopus embryos or embryos overexpressing activin at the times indicated post stage 8 and analysed by bandshift assay on the ARE. Antibodies against Smad2 (S2), XFast-1 (F1) or XFast-3 (F3) were included in the bandshift reaction where indicated, alone or with 5 µg peptide to which the antibody had been raised. ARF1 and ARF2 are indicated, as are supershifted complexes. Arrowhead indicates complex resulting from the XFast-1/3 antisera alone binding the probe; competing peptides have no effect on this complex.

View larger version (45K): [in a new window]   Fig. 3. Efficient ARF2 competition with ARF1 is explained by the high affinity SIM in XFast-3. (A) XFast-3 competes very efficiently with XFast-1 for binding activated Smads and the ARE. NIH3T3 cells were transfected with 1 µg HA-tagged XFast-1 alone or together with 0.25, 0.5, 0.75 or 1 µg HA-tagged XFast-3 or with 1 µg of XFast-3 and the same titration series of HA-tagged XFast-1. Cells were induced (or not) with TGFß1 as indicated. Whole-cell extracts were prepared and analysed for ARF1 and ARF2 by bandshift on the ARE (upper panel) or western blotted with anti-HA antibody (lower panel). ARF1 and ARF2 are indicated, as is the complex of XFast-1 with DNA. No XFast-3-DNA complex is detected. (B,C) A peptide corresponding to the XFast-3 SIM disrupts ARF1 and ARF2 much more efficiently than a peptide corresponding to the XFast-1 SIM. NIH3T3 cells were transfected with Flag-tagged XFast-1 (B) or XFast-3 (C) and incubated with TGFß1 to induce formation of ARF1 or ARF2, respectively, in a bandshift assay on the ARE. Increasing amounts (pmoles) of wild-type (wt) or mutant (mut) SIM peptides corresponding to the SIMs of XFast-3 or XFast-1 were included in the bandshift reactions as indicated.

View larger version (40K): [in a new window]   Fig. 4. XFast-3 is expressed in a narrow window at early and mid-gastrulation. (A) Total RNA was extracted from embryos at the times indicated and analysed by RNase protection using probes against XFast-1, XFast-3 or FGFR (loading control). The protected fragments are indicated, as are the undigested probes. The lane marked tRNA is a control for probe digestion. (B) XFast-1 and XFast-3 are expressed strongly in the animal cap and in the prospective mesoderm of stage 10.25 embryos. The embryos were bisected through the dorsal lip before in situ hybridisation using probes against XFast-1 or XFast-3. Specific staining is blue, and is strongest in the prospective mesoderm and ectoderm. The dark brown colour is the pigmented animal cap.

View larger version (37K): [in a new window]   Fig. 5. ARF1 and ARF2 are activated by distinct signalling molecules. (A) Single cell embryos were injected with 200 pg synthetic RNA encoding activin ßA or 1.5 ng RNA encoding derrière, Xnr1, Xnr2,VegT or BMP4. Embryos were cultured until 80 minutes post stage 8 (to detect ARF1, upper panel) or 240 minutes post stage 8 (to detect ARF2, lower panel). Nuclear extracts were prepared and assayed by bandshift assay on the ARE in the presence or absence of anti-Smad2/3 antibody. (B) Whole embryo extracts were prepared from injected embryos at 80 minutes post stage 8 (upper panels) or stage 10.5 (lower panels) and analysed by western blotting with an antibody against activated phosphorylated Smad2 (anti-P-Smad2), or Smad2/3. Xenopus embryos contain no Smad3 at this time (Howell et al., 2001). The upper band is full-length Smad2; the lower band is the spliced isoform missing exon 3 in the MH1 domain (Faure et al., 2000). (C) XFast-3 expression is inhibited by activin, Xnr1 or Xnr2. Total RNA, prepared from injected embryos at stage 10.5, was analysed by RNase protection using probes against XFast-1, XFast-3 or FGFR. Protected fragments are as indicated.

View larger version (65K): [in a new window]   Fig. 6. ARF1 and ARF2 are required for convergent extension movements of gastrulation. (A,B) Morpholinos (MOs) against either XFast-1 or XFast-3 specifically block translation in vitro and in vivo. (A) Synthetic mRNAs corresponding to native XFast-1 and XFast-3 or Flag-tagged XFast-1 and XFast-3 (as indicated) were translated in reticulocyte lysate in the presence of 0.2 mM of the indicated morpholino and [35S]-methionine. Translation products were separated by SDS-PAGE and visualised by autoradiography. (B) Nuclear extracts were prepared at the times shown from either uninjected embryos or embryos injected with 200 pg activin ßA mRNA and/or 10 ng of MOs against XFast-1, XFast-3, or both. Equal amounts of extract were analysed for the presence of ARF1 or ARF2 by bandshift using an ARE probe. Anti-Smad2/3 or anti-XFast-1 antibodies were included in the reaction as indicated to confirm the complexes as ARF1 and ARF2. The ARF1, ARF2 and supershifted (SS) complexes are indicated. The reduction of ARF1 and ARF2 in the embryos injected with MOs against XFast-1 and XFast-3 is 10- to 20-fold. (C) Depletion of ARF1, ARF2 or both has a dramatic effect on embryo development. Embryos were injected with 50 ng of control MO directed against human ß-globin, or 25 ng MO against XFast-1 or XFast-3 with 25 ng control, or 25 ng each of XFast-1 and XFast-3 MOs. Embryos were sampled when those injected with control MO reached stage 12.5 (upper panel) and stage 36 (middle panel). A medium phenotype (see Table 1) is shown in each case. Lower panel shows an in situ hybridisation on stage 36 embryos with a shh probe. Specific staining is blue. (D,E) Depletion of ARF1 or ARF2 or both partially inhibits expression of a subset of mesoendodermal genes. (D) Embryos injected with MOs as in B were cultured until those injected with control MO reached stage 10. (E) Animal caps were dissected from MO-injected embryos at stage 8 and induced with activin (40 ng/ml) until control embryos reached stage 10.25. Total RNA was prepared and analysed by RNase protection using the probes indicated. In E, the last two lanes correspond to animal caps dissected from uninjected embryos that had been treated ± activin in the presence of 5 µg/ml cycloheximide (CHX) (Howell and Hill, 1997) to indicate which genes were direct targets of the activin signalling pathway. (F) Depletion of ARF1 and ARF2 inhibits convergent extension movements of gastrulation. (a-f) Embryos injected with 50 ng control MO or 25 ng each of MOs against XFast-1 and XFast-3 were fixed when those injected with control MO reached stage 12 and whole-mount in situ hybridisation was used to detect the transcripts of Xbra, XFKH1 and XDelta1 as indicated. Specific staining is blue. Dorsal side is uppermost in all cases. (g-j) Midsagittal halves of embryos injected with control or XFast1 and XFast-3 MOs and stained for Xbra or XFKH1 transcripts. In g and i, the archenteron (A) is indicated, and the extent of the yolk plug by the white arrowheads. In h and j, the white arrow indicates dorsal blastopore lip. No archenteron has formed in these embryos. The blastocoel (B) has been squeezed as a result of some migration of mesoendoderm (black arrowheads).

View larger version (59K): [in a new window]   Fig. 7. Depletion of ARF1 and ARF2 specifically inhibits convergent extension movements in activin-induced animal caps. (A) Animal caps cut at stage 8 from embryos injected with 50 ng control MO or 25 ng each of XFast-1 and XFast-3 MOs were either untreated (left panels) or treated with activin (20 ng/ml; right panels) until control embryos reached stage 13. (B) Expression of XFast-1 or XFast-3 rescues the inhibition of activin-induced convergent extension movements in XFast-1-depleted animal caps. Animal caps cut at stage 8 from embryos injected with 25 ng XFast-3 MO and 1 ng control GFP mRNA (a,b) or 25 ng XFast-1 MO with either 1 ng control GFP mRNA (c,d), Flag-tagged XFast-1 mRNA (e) or Flag-tagged XFast-3 mRNA (f) were either untreated (left panels) or treated with activin (40 ng/ml; right panels) until control embryos reached stage 13. Note that in a,c,d-f, some of the caps have stuck together.