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doi: 10.1242/10.1242/dev.00150

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ß-Catenin/Tcf-regulated transcription prior to the midblastula transition

¹ Department of Medicine (Hematology — Oncology) and Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
² Department of Cell Biology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA

View larger version (63K): [in a new window]   Fig. 1. Dominant-negative Xtcf3 (dn-Xtcf3) blocks endogenous dorsal axis in a stage-dependent manner. (A-C) Ventralized phenotypes caused by dn-Xcf3. (A) Group I phenotype: embryos lack all dorsal-anterior structures and fail to undergo convergent extension. (B) Group II phenotype: embryos lack head structures, but maintain trunk and tail or tail alone. (C) Group III phenotype: embryos develop dorsal and anterior structures and are either normal or have small eyes and slightly reduced heads. (D) Percentage of group I, II, III embryos caused by dn-Xtcf3 mRNA injection at the four-cell stage (4cs/d2; 500 pg for each dorsal blastomere), the eight-cell stage (8cs/d4; 250 pg for each dorsal blastomere), and 16-cell stage (16cs/d4; 250 pg for each dorsal-midline blastomere). (E) Luciferase assays for embryos injected dorsally with Lef-luciferase reporter (Lef-fos) alone (Hsu et al., 1998) or with dn-Xtcf3 at the four- or the 16-cell stage. (F) Phenotypes in embryos [as in (D)] injected with ßTGR (500 pg into each dorsal blastomere at the four-cell stage). Injected embryos were cultured in normal medium (untreated) or in medium containing dexamethasone (dex) from various stages (four-cell to 128-cell) until the gastrula stage. This experiment was repeated three times (with >50 embryos per group) with similar results.

View larger version (20K): [in a new window]   Fig. 6. Pre-midblastula transition (MBT) ß-catenin/Xtcf3-dependent transcription in the regulation of dorsal development. (A) In this model, ß-catenin/Xtcf-dependent transcription begins in early cleavage stages and continues throughout pre-MBT stages (blue line). Once transcription of these pre-MBT target genes reaches a critical level (horizontal dashed line), dorsal development at post-MBT stages can proceed. If ß-catenin or Xtcf function is inhibited early (inh, red arrow) and inhibition is maintained throughout pre-MBT stages, dorsal development is blocked. However, if introduction of the inhibitor is delayed (inh, green arrow), then sufficient ß-catenin/Xtcf-dependent transcription occurs to allow dorsal development. (B) Transient inhibition of transcription with actinomycin D (ActD) until the 32-cell stage does not disrupt dorsal development because the inhibitor is reversible and thus pre-MBT transcription resumes when ActD is removed. However, if ActD is followed directly by specific inhibition of ß-catenin/Xtcf function at the 32-cell stage (inh, red arrow). at 32-cell), then dorsal development is blocked. (C) If Xtcf function is restored at the 500-cell stage (TVGR at 500 cell), then ß-catenin/Xtcf-dependent transcription resumes and reaches the threshold required for dorsal development. Thus, Xtcf function at any time during pre-MBT stages appears to be sufficient for dorsal development. TVGR can also be activated post-MBT but does not rescue dorsal development under these conditions, indicating that ß-catenin/Xtcf activity is required prior to MBT for dorsal development.

View larger version (25K): [in a new window]   Fig. 2. Inhibition of transcription in early embryos extends sensitivity to dn-Xtcf3. (A) Embryos were exposed to actinomycin D (ActD) from the two-cell stage to either the eight-cell or 16-cell stage, and dn-Xtcf3 RNA was then injected into all four dorsal blastomeres, and phenotype was scored as in Fig. 1. (B) ßTGR RNA was injected into two dorsal blastomeres at the four-cell stage. Dexamethasone (dex) was then added to embryo culture medium at the four-cell or 32-cell stage. Where indicated, embryos were cultured in ActD containing medium from the four-cell stage to the 32-cell stage and then transferred to dex-containing medium until stage ten. Phenotypes were scored at tailbud stage.

View larger version (50K): [in a new window]   Fig. 3. Polyadenylated RNA is transcribed prior to midblastula transition (MBT) in intact embryos. (A) Total RNA transcribed in early embryos was detected by injection of -[32P]UTP in two-cell embryos, which were then cultured with or without actinomycin D (ActD). RNA was prepared from various stages prior to MBT and analyzed by electrophoresis on an agarose gel, followed by autoradiography. The upper panel is an autoradiograph showing newly synthesized RNA and the lower panel shows ethidium bromide-stained ribosomal RNA from the same samples. (B) Newly synthesized polyadenylated RNA was purified by binding to oligo-dT cellulose followed by agarose gel electrophoresis and autoradiography. An equal amount of poly-A+ RNA from the 2000-cell sample was treated with RNaseA prior to loading (+RNase). Equal amounts of polyadenylated RNA were loaded in each lane, as judged by ethidium bromide staining (not shown).

View larger version (52K): [in a new window]   Fig. 4. Pre-midblastula transition (MBT) transcription of Xnr5 and Xnr6 is regulated by ß-catenin and Xtcf3 in dorsal blastomeres. (A) Zygotic expression of Xnr5 and Xnr6 is detectable by RT-PCR as early as the 256-cell stage whereas siamois expression begins at the 4000-cell stage. Ornithine decarboxylase (ODC), a maternally expressed gene that does not increase significantly at MBT, was used as a loading control. `No-RT' indicates control lacking reverse transcriptase. (B) Regulation of pre-MBT transcription by ß-catenin: embryos were injected ventrally with ß-catenin mRNA (500 pg), treated at the 32-cell stage with LiCl (0.3 M for 10 minutes), or injected dorsally with either dnXtcf3 mRNA (500 pg) or morpholino antisense oligonucleotide against ß-catenin (10 ng). Embryos were harvested at the 1000-cell stage and analyzed by RT-PCR for Xnr5 and Xnr6 expression (FGFR: FGF receptor was a loading control). (C) Pre-MBT transcription of Xnr5 and Xnr6 is localized to dorsal blastomeres. Embryos were dissected into dorsal and ventral halves at the 500-cell stage, RNA was isolated from each half, and Xnr5 and Xnr6 expression was assessed by RT-PCR as in panels (A) and (B). Control whole embryo RNA is shown in lane 1. (D) RNA polymerase II-dependent pre-MBT transcription of Xnr5 and Xnr6: embryos were treated with actinomycin D (ActD) from the two-cell stage, injected with the RNA polymerase II-specific inhibitors -amanitin or 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB) at the one-cell stage, or treated with LiCl as in (B). Embryos were harvested at the 1000-cell stage for analysis of Xnr5 and Xnr6 expression as above.

View larger version (30K): [in a new window]   Fig. 5. Tcf-dependent transcription is required prior to midblastula transition (MBT) for dorsal development. (A) To antagonize ß-catenin function and thereby block dorsal development, axin mRNA (2.5 ng) was injected into both dorsal blastomeres at the four-cell stage, as described (Zeng et al., 1997). To rescue dorsal development, hormone-inducible Xtcf3 (TVGR; 0.5 pg) was co-injected and Dexamethasone (dex) was then added at the four-cell, 500-cell stage, MBT (the 4000-cell stage), or late blastula (stage nine). Phenotypes were scored at tailbud stage. (B) Luciferase assays for embryos injected with Lef-fos (as in Fig. 1E) and TVGR (0.5 pg) into the animal pole at the two-cell stage. Injected embryos were cultured in normal medium (no dex), or dex was added at the 128-cell stage (dex, 128cs) or at MBT (dex, MBT). Embryos were harvested every 30 minutes after MBT and luciferase activity was measured. (C) Activation of Xtcf3 induces pre-MBT transcription in ventralized embryos: Axin mRNA was injected into dorsal blastomeres at the four-cell stage alone or with TVGR, as in (A). Injected embryos were cultured in normal medium (no Dex) or dex was added at the four-cell stage (Dex, 4cs). Embryos were harvested at the 1000-cell stage and analyzed for Xnr5 and Xnr6 expression. (EF1- was the loading control). (D) Activation of Xtcf3 rescues expression of Siamois and Xnr3 only if hormone is added before MBT, although activation after MBT can still rescue Xnr6 transcription: injection and manipulation were performed as described in (A). Embryos were harvested at early gastrula stage (stage ten+) and analyzed by RT-PCR for Siamois, Xnr3 and Xnr6 expression (EF1- was a loading control). Lane 1 represents uninjected gastrula-stage embryos.