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In early Xenopus embryos, the prototypical XFast-1/Smad2/Smad4 complex ARF1 is induced at the Mix.2 ARE by activin overexpression. We have characterised ARF2, a related, but much more abundant, complex formed during gastrulation in response to endogenous TGFbeta family members and we have identified a novel Fast family member, XFast-3, as its transcription factor component. Endogenous ARF2 efficiently competes out ARF1 at early gastrulation, due to the ability of XFast-3 to interact with activated Smads with much higher affinity than XFast-1. We demonstrate that ARF1 and ARF2 are activated by distinct TGFbeta family members. Using morpholino antisense oligonucleotides to deplete levels of the constituent transcription factors XFast-1 and XFast-3 specifically, we demonstrate an important role for ARF1 and ARF2 in early Xenopus embryos in controlling the convergent extension movements of gastrulation.
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.
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 βA 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 β-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).