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The transcription factor FAST-1 has recently been shown to play a key role in the specification of mesoderm by TGF beta superfamily signals in the early Xenopus embryo. We have cloned Fast1, a mouse homologue of Xenopus FAST-1, and characterized its expression during embryogenesis and function in activin/TGF beta signal transduction. In vitro, Fast1 associates with Smads in response to an activin/TGF beta signal to form a complex that recognizes the Xenopus activin responsive element (ARE) targeted by Xenopus FAST-1. In intact cells, introduction of Fast1 confers activin/TGF beta regulation of an ARE-luciferase reporter. In embryos, Fast1 is expressed predominantly throughout the epiblast before gastrulation and declines as development progresses. We propose that mouse Fast1, like Xenopus FAST-1, mediates TGF beta superfamily signals specifying developmental fate during early embryogenesis.
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10349617
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Fig. 2. Participation of Fast1 in ARF complex. (A) cDNA encoding Fast1 was transfected into 293 cells in the presence or absence of activated Type I activin receptor (ActRIB*), cells were lysed and assayed for ARF activity by EMSA. (B) cDNA encoding Myc-tagged Fast1 was transfected into 293T cells in the presence of ActRIB* and epitope tagged Smad2 and Smad4. Lysates were incubated with anti-epitope antibodies indicated and assayed by EMSA. ssARF (supershifted ARF) denotes ARF complex shifted in mobility by binding of anti-epitope antibodies to tagged ARF components. No supershifts were observed when antibodies were added in the absence of epitope tagged Smads or Fast1 (not shown).
Fig. 3. Regulated co-precipitation of Fast1 with Smad2 and Smad4. Epitope tagged Fast1 or Smads were expressed by mRNA microinjection into Xenopus embryos in the presence or absence of activin. Embryos were harvested and immunoprecipitated with the anti-FLAG antibody, Fast1 co-precipitation was then detected by anti-myc tag Western blotting. Bottom panels confirm equal expression of tagged Fast1 in whole cell lysates. (A) Activin stimulates co-precipitation of Smad2 with Fast1, but BMP4 does not stimulate Smad1 co-precipitation of Fast1. (B) Activin stimulates co-precipitation of Fast1 and Smad4.
Fig. 6. Expression of Fast1 during early mouse embryogenesis. Localization of Fast1 and murine T transcripts in 6.0–8.5 dpc embryos, as revealed by whole-mount and section in situ hybridization. (A) Section through a 6.0 dpc pre-gastrulation stage embryo showing Fast1 expression in the epiblast, but not in the extra-embryonic ectoderm. Solid line shows the junction between the two regions. (B) Transverse section of an embryo at the same stage, at the level indicated in (A), showing Fast1 expression throughout the epiblast and probably the embryonic endoderm. (C-D) 6.5 dpc embryo from the same litter as (E,F) probed for Fast1. Fast1 transcripts are still present throughout the embryo, which has initiated gastrulation, but not in the extra-embryonic ectoderm. (E,F) T (Brachyury) expression is detected in the primitive streak at the posterior of the embryo. (G) At 7.5 dpc, Fast1 is ubiquitously expressed in the developing embryo, while T transcripts are localized to the notochord and primitive streak (H). (I,J) At 8.0 and 8.5 dpc, Fast1 expression is strongest in the heart and allantois, while low levels are visible in the notochord and posteriormesoderm. Branchial arch (b); heart (h); epiblast (ep); embryonic endoderm (en); extra-embryonic ectoderm (ee); allantois (al); neural folds (nf); presomitic mesoderm (p).
Fig. 7. Expression of Fast1 and endogenous ARE-luciferase responsiveness in P19 cells. (A) Northern analysis measuring Fast1 mRNA levels in NIH 3T3 cells (lane 1) and P19 cells (lane 2). (B) RT-PCR measuring Fast1 mRNA levels in NIH 3T3 cell (lane 1) and P19 cells (lane 2). As a control, RT-PCR was done on cellular RNA in the absence of reverse transcriptase (lane 3). A water blank is shown in lane 4. Levels of GAPDH were measured for both Northern analysis and RT-PCR as an internal loading control. C) Endogenous ARE responsiveness of P19 cells versus NIH 3T3 cells; P19 cells were transfected with the multimerized ARE Luciferase reporter construct A3, and or ACTRIB*, and Fast1. The background is defined as the Luciferase activity from cells transfected with the ARE-Luciferase reporter construct alone. The results are of a representative experiment, performed in triplicate.
