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Figure 1. General structure of expression and reporter plasmids. A: Expression plasmid with ORF of pitx3 bicistronically linked to eGFP. B: Reporter plasmid with the reporter gene DsRed-express driven by the tested promoter, cloned in opposite orientation from the transfection control gene HcRed1 driven constitutively by CMV.
Figure 1. Correlation between the pitx3 and GFP proteins. The proteins were assayed by Western blotting and the amount of each protein was assessed as the optical integrated density value of the respective band. A: Figure 2. Regression analysis to correlate the levels of pitx3 and GFP proteins in cells transfected with different concentrations of expression vector. B: Ratios between the levels of pitx3 and GFP protein in cells transfected with a set concentration of expression vector and analyzed at 24, 36, and 48 hr post-transfection. There is no statistically significant difference between time points of this range.
Figure 3. Correlation between GFP protein and GFP fluorescence. The GFP protein levels were determined by Western blotting and evaluated as the optical density of the band on the blot. A percentage of the total cells was used to detect the fluorescence using flow cytometry. A: Regression analysis to correlate GFP protein levels and GFP fluorescence in cells transfected with four decreasing concentrations of expression vector by 1.3-fold. B: Linear regression between the GFP protein and fluorescence in cells transfected with equal concentrations of expression vector and evaluated at three different times post-transfection.
Figure 4. Flow cytometer setup to detect the three fluors in the new reporter assay. A: Gate setup was arranged to minimize spectral overlap and background. For eGFP in FL1 for cells transfected with the IRES-GFP, control vector relegates GFP counts to the right of the vertical line in quadrant G4. B: Cells transfected with CMV-HcRed control plasmid, recorded in FL5 and gated for HcRed1 expression, relegate HcRed counts to above the horizontal line in quadrant G1. C: Gated population of 10,000 cells expressing eGFP, HcRed1, and DsRed (Ds Red, indicative of promoter activity, is counted in quadrant G2). D: Histogram of DsRed output in FL2 for the population of eGFP and HcRed co-expressing cells.
FIGURE 5. Pitx3 represses the Th (tyrosine hydroxylase) promoter. The Th reporter (plasmid CMV-HcRed/Th-DsRed) was tested, under conditions of presence or absence of forskolin, to see how it responded to vectors for pitx3 absent (IRES-GFP), pitx3 protein (pitx3-IRES-GFP), or pitx3mutHD expressing a homeodomain mutant (pitx3mutHD-IRES-GFP). ANOVA demonstrated that statistically significant differences existed. A post hoc Tukey test determined specifically which comparisons were significant and P values are reported above. ns, no significant difference
Figure 6. The Th (Tyrosine hydroxylase) promoter binding motif for pitx3 is important for repression. Wild-type and mutant Th reporters (wt: CMV-HcRed/Th-DsRed; mutant: CMV-HcRed/Thmut-350-DsRed) were tested under conditions of the presence or absence of pitx3 (absent: plasmid IRES-GFP; present: plasmid pitx3-IRES-GFP). ANOVA demonstrated that statistically significant differences existed. A post hoc Tukey test determined specifically which comparisons were significant and P values are reported above. ns, no significant difference.
Figure 7. Th promoter is activated in linear fashion by pitx3. Transcription factor activity was assessed in cells expressing all three fluors by plotting the normalized DsRed output to the GFP input and determining the generated trend line.
Figure 8. lhx1 promoter activation by pitx3. Vectors for pitx3 absent (IRES-GFP), pitx3 protein (pitx3-IRES-GFP), or pitx3mutHD expressing a homeodomain mutant (pitx3mutHD-IRES-GFP) were tested for their ability to activate a wild-type lhx1wt promoter-driven DsRed reporter (CMV-HcRed/lhx1-DsRed) and a pitx3 binding motif mutant lhx11mut-709 promoter-driven DsRed reporter (CMV-HcRed/lhx1mut-709-DsRed). ANOVA demonstrated that statistically significant differences existed. A post hoc Tukey test determined specifically which comparisons were significant and P values are reported above. ns, no significant difference.
Figure 9. The xnr5 promoter is repressed by pitx3. Wild-type and mutant xnr5 reporters (wt: CMV-HcRed/xnr5-DsRed; mutant: CMV-HcRed/xnr5mut-3-94-DsRed) were tested under conditions of the presence or absence of pitx3 (absent: plasmid IRES-GFP; present: plasmid pitx3-IRES-GFP), as well as in the presence or absence of an activator, vegT, or a pitx3 homeodomain mutant pitx3mutHD-IRES-GFP. ANOVA demonstrated that statistically significant differences existed. A post hoc Tukey test determined specifically which comparisons were significant and P values are reported above. ns, no significant difference.
Figure 10. Differential gene expression at early gastrula stages and during laterality. A,A': A GFP reporter expresses in the dorsal lip when the pitx3 binding site in the lhx1 promoter is intact (A), but is down-regulated when mutated (split exposure image in A'; both fluorescence images exposed 19 sec). B–D: Vegetal view of embryos injected with control-morpholino (CMO) display wild-type gene expression patterns. B'–D': But pitx3-morpholino (P3MO) affects gene expression during the beginning of gastrulation. Dorsal lip is oriented to the top. Note the lateral depression of laterallhx1 and t (arrows in B' and D', respectively) and expansion of gsc expression in P3MO embryos and knockdown embryos (compare C and C', arrows). E–G: Genes participating in the laterality signal cascade are normally expressed in leftlateral plate mesoderm (arrows). E'–G': In morphants where pitx3 is knocked down, expression of xnr5 downstream targets xnr1, lefty, and pitx2 is decreased or absent in the leftlateral plate mesoderm (arrows).
