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Fig. 1. Embryos transgenic for [-4694 to +436] express eGFP in a pattern that recapitulates endogenous geminin expression. Embryos were analyzed by in situ hybridization for eGFP (A C) or Xgem (D F) at st. 11.5 (A, D), st. 23 (B, E) and st. 35 (C, F). (A, D) Lateral views, dorsal up and anterior facing right. (B, C, E, F) Lateral views, dorsal up. (B, E) Embryos are curved with anterior structures facing out.
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Fig. 2. 5V deletion series identifies two sequence domains that regulate dorsal-specific expression. Transgenic embryos carrying the constructs schematized in A were analyzed by in situ hybridization at st. 11.5 for the pattern of eGFP expression. (B, D, F, H, J) Side views, posterior facing right, and dorsal up. (C, E, G, I, K) Posterior views with dorsal up. (C, E, G, I, K) Numbers represent the fraction of total embryos carrying the indicated construct that displayed the expression pattern shown.
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Fig. 3. Internal deletion series confirms the necessity of two 5V regulatory domains. Transgenic embryos carrying the constructs schematized in A were analyzed by in situ hybridization at st. 11.5 for the pattern of reporter expression. (B, D, F, H, J) Side views with posterior to the right and dorsal up. (C, E, G, I, K) Posterior views with dorsal up. (B, J, K) In situ for eGFP. (H, I) In situ for firefly luciferase. (C, E, G, I, K) Numbers represent the fraction of total embryos carrying the indicated construct that displayed the expression pattern shown.
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Fig. 4. Sufficiency of sequence domains to drive or restore dorsal-specific expression. Transgenic embryos carrying the constructs schematized in A were analyzed by in situ hybridization at st. 11.5 for the pattern of reporter expression. [-3783 to -2920] + [-2425 to -1580] and [-3783 to -2920] + [-2289 to -2020] were subcloned into pRAREsCMV. D[-2404 to -1611] + [-2289 to ô´•2020] and D[-2404 to -1611] + [-2025 to -1775] were subcloned into pGL3basic. (B, D, F, H) Side views with posterior to the right and dorsal up. (C, E, G, I) Posterior views with dorsal up. (B–E) In situ for eGFP. (F–I) In situ for firefly luciferase. (C, E, G, I) Numbers represent the fraction of total embryos carrying the indicated construct that displayed the expression pattern shown.
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Fig. 5. Mutation of putative Tcf- and Vent-binding sites in the geminin 5V regulatory region alters reporter expression pattern in transgenic gastrulae. Using the construct schematized in A, consensus binding sites for Vent and Tcf at the positions shown were mutated (see Materials and methods). Sites are numbered relative to the transcription start site. (B M) Transgenic embryos carrying the non-mutated or mutated constructs were analyzed at early (st. 10.5; B G) and mid-gastrulation (st. 11.5; H M) by in situ hybridization for eGFP expression. (B, C, H, I) Construct with non-mutated sites. (D, E, J, K) Construct with mutated XVent1 and XVent2 sites. (F, G, L, M) Construct with all three Tcf sites mutated. (B, D, F, H, J, L) Side views with posterior to right and dorsal up. (C, E, G, I, K, M) Posterior views with dorsal up. (C, E, G, I, K, M) Numbers represent the fraction of total embryos carrying the indicated construct that displayed the expression pattern shown.
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Fig. 6. geminin regulatory sequences respond to the Wnt and BMP signaling pathways in the gastrula embryo. (A D) In situ hybridization for eGFP in embryos transgenic for [-4694 to +436] plasmid. Animal hemisphere views of late gastrula embryos (st. 12), dorsal up. Embryos were injected in one cell at the two cell stage (left bilateral half in the images shown) with bmp4 (B), beta-catenin (C), or noggin (D) mRNA. (A) shows an uninjected sibling embryo. (E) Luciferase assays of embryos injected with pGL3basic or this vector containing 5.1 kb of geminin 5V regulatory sequences. Samples were co-injected with mRNAs as indicated. Normalized values (Firefly to Renilla) for three pools of embryos per sample were averaged. Representative results are shown. Error bars represent standard errors. (F) In situ hybridization for Xgem at st. 11.5. (F) Dorsal views with posterior down. (K) Side view with posterior to the right and dorsal up. (F) uninjected embryo (G) Injected with beta-catenin morpholino oligonucleotide into 1 cell at the 2-cell stage. (H K) Injected with mRNA for Xvent1 (H), Xvent2 (I), VPXvent-1 (J), or VPXvent-2 (K) into one A-tier blastomere at the 32-cell stage. (G K) Blue X-gal staining indicates site of injection. (B D and G) mRNA delivery was not restricted to a small ectodermal domain, and axial patterning of the injected embryos was broadly affected.
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Fig. 7. Conservation of Regulatory Domains. (A) The 5.1 kb of hgem 5V sequence is aligned to 6300 bp of Xgem 5V sequence using WU-BLAST (http:// blast.wustl.edu) (parameters: W = 5, M = 5, N = 3, Q = 10, R = 10). Sequences are represented by gray bars. In hgem, the two regulatory domains are represented by dark gray bars. Aligned sequences between hgem and Xgem are represented by medium-gray bars and linked by green lines marking the ends of the alignments. Putative binding sites for Tcf, XVent1 and XVent2 are labeled by red, green and blue vertical lines, respectively. These putative sites are determined by scanning both sequences against scoring matrices of these transcription factors using the program PATSER (http://ural.wustl.edu) and using cutoff values determined by the program. Locations of lines (above or below the sequences) indicate the orientation of the sites (on forward or reverse strand respectively). Lengths of lines are proportional to the scores of the sites which indicate the strength of the sites. (B, C) Transgenic embryos carrying 5 kb of upstream sequence from Xgem cloned into pRAREeGFP were analyzed at st 11.5 by in situ hybridization for eGFP. (B) side view with posterior to the right and dorsal up. (C) posterior view with dorsal up. Numbers indicate sequence location relative to the transcription start site.
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Fig. 8. Model of regulation of early geminin expression. (A) Early gastrula:. Nuclear-localized Beta-catenin is active dorsally. Our data demonstrates that Beta-catenin can activate Xgem and the geminin 5V sequences and that Tcf sites in the geminin regulatory sequences are necessary for geminin expression in dorsal ectoderm at early gastrulation. (B) Mid gastrula: geminin can activate its own transcription. BMP4 activates the transcriptional repressors XVent1 and XVent2. Our data demonstrates that XVent1 and XVent2 sites in the geminin regulatory sequences are necessary to restrict geminin expression to dorsal tissues at mid-gastrulation. We also found that Xgem or geminin regulatory sequences responded negatively to BMP signaling. Schematics are side views of st. 10.5 (A) and st. 11.5 (B) Xenopus gastrulae with posterior facing right and dorsal up.
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