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Figure 1. Schematic representation of the HpOtxE promoter (−461 to +180 bp, open rectangle) and the SpSpec2a core promoter (−66 to +23 bp, gray rectangle) used in this study to express the CFP or YFP reporter gene. The HpOtxE core promoter (−66 to +23 bp, open rectangle) was replaced with the SpSpec2a core promoter to test the functional interactions between the HpOtxE upstream sequence and the SpSpec2a core promoter sequence. The vertical rectangles on the promoters are putative binding sites for the GATA factor (−360 to −355 bp and −250 to −245 bp, black rectangles), the Otx factor (−294 to −289 bp, open rectangle) and USF (E-box, ∼−66 to −61 bp, hatched rectangle). The nucleotide sequences of the HpOtxE and SpSpec2a core promoters are shown at the top and the bottom, respectively. The transcriptional initiation site is highlighted with large bold letters and an arrow, the E-box with a hatched rectangle and the sequences that are mutated in this study with large bold letters.
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Figure 2. Classification of the expression patterns of CFP/YFP reporter genes in transfected embryos. The expression of these two reporters were examined simultaneously in the same embryos using a fluorescence microscope (×200 magnification) at the blastula, gastrula and pluteus stages. Arbitrarily selected representative examples are shown as overlaid fluorescent and bright-field images. The CFP/YFP reporter plasmids transfected in these experiments are as follows: pM4694/pM4706 (A, C), pM4705/pM4692 (B), pM4947/pM4942 (D), pM4725/pM4692 (E), pM4705/pM4706 (F), pM7070/pM4706 (G, I), pM4694/pM7071 (H), pM4725/pM7073 (J), pM4694/pM7067 (K) and pM7070/pM7073 (L). Cells that express CFP or YFP are shown in green and red, respectively, whereas those that express both are shown in yellow. The expression patterns were classified into four groups as indicated: Profile #1, green and red signals completely overlapped; Profile #2, the signals partially overlapped or did not overlap; Profile #3, one signal was included within the boundary of the other and Profile #4, only one signal was observed.
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Figure 3. Verification of the system for monitoring the functional interactions between the upstream and the core promoter sequences. In each panel, the schematic on the left indicates the reporter plasmids introduced into fertilized eggs. (A) Expression of CFP/YFP reporter genes driven by the same promoters. Three sets of CFP/YFP reporter plasmids were used: pM4705/pM4692, driven by the wild-type HpOtxE promoter; pM4725/pM4731, driven by the wild-type SpSpec2a promoter and pM4694/pM4706, driven by a mutated HpOtxE promoter bearing the TATAAA sequence instead of the TATTCA sequence (OtxEM; changed nucleotides are shown in red). The expression of these reporter genes were examined with fluorescence microscopy at the blastula, early gastrula, mid gastrula, late gastrula and pluteus stages, as indicated at the top. The number of embryos showing the expression pattern corresponding to each profile (defined in Figure 2) is summarized in the right panel. The vertical and horizontal axes indicate the number of embryos and expression pattern profiles, respectively. (B) Expression of CFP/YFP reporter genes driven by promoters containing or not containing the upstream sequence. (Upper panel) The reporter plasmids used were pM4705/pM4915 (I) or pM4914/pM4692 (II), which are driven by the wild-type HpOtxE promoter and its core promoter connected to CFP and YFP, respectively (I), or vice versa (II). The expression of the reporter genes was examined with fluorescence microscopy at different developmental stages, as indicated at the top. The number of embryos showing the expression patterns corresponding to each profile is summarized in the right panel as described in A. The color of the bars representing profile #4 reflect the signal that was observed (CFP, green; YFP, red). (Lower panel) Two CFP/YFP reporter plasmids were used, pM4725/pM4917 (I) and pM4916/pM4731 (II), which are driven by the wild-type SpSpec2a core promoter fused or not fused with the HpOtxE upstream sequence and connected to the complementary patterns of CFP and YFP. The number of embryos showing the expression patterns corresponding to each profile is summarized in the right panel as described above.
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Figure 4. The effect of the TATA element on core promoter usage by the upstream sequence. In each panel, the schematic on the left indicates the reporter plasmids introduced into fertilized eggs. I and II refer to which reporter (CFP or YFP) was connected to each promoter, as indicated by the boxes marked C and Y. The number of embryos showing the expression pattern corresponding to each profile is summarized in the panel to the right of each schematic, as described in Figure 3. (A) The CFP/YFP reporter plasmids used were pM4705/pM4731 (I) and pM4725/pM4692 (II), which are driven by the wild-type HpOtxE promoter connected to CFP and the wild-type SpSpec2a core promoter fused with the HpOtxE upstream sequence. The bars representing profile #3 are shown as rectangles composed of two triangles; the left upper triangle corresponds to the signal that was observed in the broader region of the embryo (CFP; green, YFP; red). (B) The reporter plasmids used were pM4705/pM4734 (I) and pM4728/pM4692 (II), which are driven by the wild-type HpOtxE promoter and the mutated SpSpec2a core promoter (Spec2aM; the AATAATA sequence was removed as indicated with double red lines) fused with the HpOtxE upstream sequence. (C) The reporter plasmids were pM4705/pM4706 (I) and pM4694/pM4692 (II), which are driven by the wild-type HpOtxE promoter and the mutated HpOtxE promoter (OtxEM as described in Figure 3A). (D). Each set of the two CFP/YFP reporter plasmids, i.e. pM4725/pM4734 (I) or pM4728/pM4731 (II), which are driven by the wild-type and the mutated SpSpec2a core promoter (Spec2aM as described in B) fused with the HpOtxE upstream sequence.
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Figure 5. The effect of the four putative transcription factor binding sites on spatiotemporal expression of the TATA-less and TATA-containing HpOtxE promoters. In each panel, the schematic on the left indicates the reporter plasmids introduced into fertilized eggs. I and II refer to the particular combinations of reporter (CFP or YFP) and promoter, as indicated by the boxes marked C and Y. The number of embryos with each expression pattern profile is summarized in the panel to the right of each schematic. (A) (First panel) The plasmids used were pM4705/pM4938 (I) and pM4937/pM4692 (II), driven by the HpOtxE promoter fused with the intact or mutated HpOtxE upstream sequence within the putative GATA factor binding site #1 (−360 to −355 bp). (Second panel) Similar analysis using pM4940 (YFP) and pM4939 (CFP), in which the HpOtxE upstream sequence was mutated within the putative Otx factor binding site (−294 to −289 bp), instead of pM4938 (YFP) and pM4937 (CFP) in the first panel. (Third panel) Analysis using pM4942 (YFP) and pM4941 (CFP), in which the HpOtxE upstream sequence was mutated within the putative GATA factor binding site #2 (−250 to −245 bp). (Fourth panel) Analysis using pM4783 (YFP) and pM4782 (CFP), in which the HpOtxE upstream sequence was mutated within the E-box (−66 to −61 bp). (B) (First panel) pM4694/pM7065 (I) or pM7062/pM4706 (II), which are driven by the mutated HpOtxE core promoter (OtxEM as described in Figure 3A) fused with the intact or mutated HpOtxE upstream sequence within the putative GATA factor binding site #1. (Second panel) pM7066 (YFP) and pM7063 (CFP), in which the HpOtxE upstream sequence was mutated within the putative Otx factor binding site. (Third panel) pM7067 (YFP) and pM7064 (CFP), in which the HpOtxE upstream sequence was mutated within the putative GATA factor binding site #2. (Fourth panel) pM4781 (YFP) and pM4780 (CFP), the HpOtxE upstream sequence was mutated within the E-box.
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Figure 6. Comparison of the functional interactions between the different upstream sequences and the TATA element within the HpOtxE core promoter. In each panel, the schematic on the left indicates the reporter plasmids introduced into fertilized eggs. I and II refer to which reporter (CFP or YFP) was connected to each promoter, as indicated by the boxes marked C and Y. The number of embryos showing the expression pattern corresponding to each profile is summarized in the panel to the right of each schematic. (First panel) pM4705/pM4706 (I) or pM4694/pM4692 (II), which are driven by the wild-type and mutated HpOtxE core promoter (OtxEM as described in Figure 3A) fused with the intact HpOtxE upstream sequence. (Second panel) pM4937/pM7065 (I) or pM7062/pM4938 (II), in which the HpOtxE upstream sequence was mutated within the putative GATA factor binding site #1. (Third panel) pM4939/pM7066 (I) or pM7063/pM4940 (II), in which the HpOtxE upstream sequence was mutated within the putative Otx factor binding site. (Fourth panel) pM4941/pM7067 (I) or pM7064/pM4942 (II), in which the HpOtxE upstream sequence was mutated within the putative GATA factor binding site #2. (Fifth panel) pM4782/pM4781 (I) or pM4780/pM4783 (II), in which the HpOtxE upstream sequence was mutated within the E-box.
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Figure 7. Comparison of functional interactions between the different upstream sequences and the HpOtxE/SpSpec2a core promoters. In each panel, the schematic on the left indicates the reporter plasmids introduced into fertilized eggs. I and II refer to which reporter (CFP or YFP) was connected to each promoter, as indicated by the boxes marked C and Y. The number of embryos showing the expression pattern corresponding to each profile is summarized in the panel to the right of each schematic. (First panel) pM4705/pM4731 (I) or pM4725/pM4692 (II), which are driven by the wild-type HpOtxE promoter and the wild-type SpSpec2a core promoter fused with the intact HpOtxE upstream sequence. (Second panel) pM4937/pM4944 (I) or pM4943/pM4938 (II), in which the HpOtxE upstream sequence was mutated within the putative GATA factor binding site #1. (Third panel) pM4939/pM4946 (I) or pM4945/pM4940 (II), in which the HpOtxE upstream sequence was mutated within the putative Otx factor binding site. (Fourth panel) pM4941/pM4948 (I) or pM4947/pM4942 (II), in which the HpOtxE upstream sequence was mutated within the putative GATA factor binding site #2. (Fifth panel) pM4782/pM4789 (I) or pM4788/pM4783 (II), in which the HpOtxE upstream sequence was mutated within the E-box.
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Figure 8. Developmental changes in the expression of CFP/YFP reporter genes driven by the same or different pairs of promoters within single embryos immobilized in solidified agar. (A–D). The same set of the CFP/YFP reporter plasmids as used in Figure 3A (bottom panel), pM4705/pM4692, was introduced into fertilized eggs. The expression of these two reporters was monitored semicontinuously within the same embryos using fluorescence microscopy (×200 magnification). Photographs were taken 21 h (A), 25 h (B), 27 h (C) and 31 h (D) after fertilization, and are shown here as overlaid fluorescent and bright-field images as described in Figure 2. (E–H). The same set of CFP/YFP reporter plasmids used in Figure 7 (Third panel, II), pM4945/pM4940, was introduced into fertilized eggs. The expression of these two reporters was monitored as described above. Photographs were taken at 21 h (E), 24 h (F), 26 h (G) and 29 h (H) after fertilization. (I–L). The same set of CFP/YFP reporter plasmids used in Figure 4A (I), pM4705/pM4731, was introduced into fertilized eggs. The expression of these two reporters were monitored as described above. Photographs were taken at 21 h (I), 25 h (J), 27 h (K) and 29 h (L) after fertilization.
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