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Figure 1. Early HDAC mRNA injection induces heterotaxia. (A) Xenopus HDAC mRNA expression pattern is shown by in situ hybridization with early Xenopus embryos. HDAC mRNA presents a symmetric expression pattern by animal cells at the stage 3 (4 cells) (a,b,c), at the stage 5 (16 cells) (d,e,f) and at the stage 7 (64 cells) (g,h,i). in situ reaction control with no secondary (j, animal view) or no probe added to the reaction (k, animal view; l, vegetal view). (B) Embryos injected with HDAC DN on the ventral or dorsal right blastomeres presented significant levels of heterotaxia (VR 20% heterotaxia p < 0.01, n = 55; DR 19% heteroatxia p < 0.01, n = 42; VL 7% heterotaxia p = 0.3 n = 55; DL 2% heterotaxia p = 0.7). Embryos injected with HDAC WT on the ventral left side presented significant levels of heteroatxia (VL 19% heterotaxia p < 0.01, n = 68; VR 4% heterotaxia p = 0.8, n = 49; DL 4% heteroataxia p = 0.8, n = 44; DR 2% heterotaxia p = 0.5, n = 50). (B`) Schematic showing the injected blastomere at the 4 cell stage. (I) Wild type phenotype showing the gut coil to left (yellow arrow), the gall bladder on the right (green arrow) and the heart loop on right (red line). (II) Situs inversus phenotype where all three organs analyzed are found inverted. (III) Heterotaxic phenotype showing the heart loop to the left (red arrow).
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Figure 4. Xenopus Mad3 mRNA and protein are present in early embryos. (A) Mad3 mRNA is expressed by animal cells in a symmetric pattern at the stage 3 (4 cells) (I,II,III), at the stage 5 (16 cells) (IV,V,VI) and at the stage 7 (64 cells) (VII,VIII,IX). Figure X: in situ reaction control, animal view (no secondary antibody added to the reaction); XI and XII: no probe added to the reaction, animal and vegetal view, respectively. (B) Immunoblotting using stage 7 (64 cells) whole embryo lysate with an anti-Mad3 antibody. Lane 1: Whole lysate from stage 7 embryos. Anti-Mad3 recognizes a band running at 30 kDa. Lane 2: Immunoblotting control with no primary antibody added to the membrane. Lanes 3 and 4: Whole protein lysate from non injected (lane 3) or Mad3WT-flag injected embryos (lane 4) were used to detect exogenous Mad3.
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Figure 5. Mad3 is a new left-right determinant. (A) Single ventral left or ventral right blastomeres were injected with EngMad3 or Vp16Mad3 constructs and organ placement was scored at stage 45. Significant levels of heterotaxia were observed when embryos were injected with EngMad3 (left: 21%, n = 96, p < 0.001; right: 12%, n = 91, p = 0.005; control: Eng) or with Vp16Mad3 on the right side (17% n = 90, p < 0.001; 6% n = 127, p = 0.1, control: Vp16). (B) Embryos injected with EngMad3 or Vp16Mad3 were processed for in situ hybridization at stage 21 with a XNr-1 probe. XNr-1 misexpression was correlated with Vp16Mad3 injection (right injected embryos: 19% bilateral expression, n = 117; left injected: 5% bilateral expression, n = 105) and EngMad3 (left injected: 75% no expression, n = 74; right injected: 21% no expression, n = 93) if compared to control embryos (uninjected, 10% no expression, n = 103). (B') XNr-1 expression pattern characterized in EngMad3 or Vp16Mad3 injected embryos. I- Left expression indicated by the white arrow; II-right expression indicated by the gray arrow; III- Bilateral XNr-1 expression indicated by the two blue arrows; IV- Absence of XNr-1 expression as indicated by the two black arrows.
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Figure 7. HDAC activity is required during LR development. (A) In this study, we found that early HDAC activity between stages 1-7 is important to proper set the pattern of expression of XNr-1 at stage 21. In addition, we also show that HDAC activity and Mad3 are important on the right side of the embryo to set XNr-1's expression and that Mad's biological activity is dependent on 5HT. Despite HDAC and Mad3 mRNAs present a symmetric pattern of expression, one theory is that because 5HT is asymmetrically distributed in the right blastomeres at stage 7 (red), 5HT binding on Mad3 (blue) may recruit HDAC activity (orange) to the intronic region of XNr-1 on the right of the embryo. HDAC activity, at early developmental stages, may be important to decrease the levels of acetylation of histone H3 and H4 on the intronic region of Nr1. (B) At stage 21, low levels of acetylation on histones (H3 and H4), set by early HDAC activity, would contribute to repress the expression of Nr1, that will be expressed only on the left side of the embryo (blue box). V-ventral; D-dorsal; R-right; L-left.
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Figure 2. HDACs exert stage specific effect on left-right patterning. (A) Schematic showing the experimental procedure for NaB treatments. Embryos were exposed to 100 mM NaB from stage 1 to 7 (solid black line) and the drug was washed out (dashed black line). Embryos were collected at stage 7 for anti-acetyl H4 western blotting or were allowed to develop in 0.1X MMR until stage 45 to score organ placement or until stage 21 for ChIP experiments. (B) 100 mM NaB treatment exerts stage specific effects on left-right development. Embryos that were exposed to NaB between stage 1-7 presented significant levels of heterotaxia (100 mM NaB: 22.5%, p < 0.001). NaB exposure after stage 7 does not affect left-right development (stage 7-8 and stage 8-9). (C) NaB treatment led to a significant increase in the H4 acetylation levels. Embryos exposed to NaB from stage 1 to 7 presented 1.8 fold increase, stage 7-8 presented 2.3 fold increase and stage 8-9 presented 2.3 fold increase on the levels of acetylated histone H4 when compared to controls. (* p < 0.05).
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Figure 3. HDAC inhibition leads to increased levels of acetylated histones and H3K4me2 on the XNr-1 gene. (A) Schematic showing the structure of the Xenopus Nr-1 gene. Light gray boxes represent the protein-coding and the dark gray box represents the promoter region (adapted from [53]). Intronic regions 1 and 2 are indicated. Red and blue arrows represent the primer set used for qPCR reaction for the promoter and intronic region, respectively. (A1) The red lines indicate the sequence of the XNr-1 promoter region used to design the primer set for qPCR reaction. (A2) The regions underlined show the sequence used for primer set design. In purple are highlighted the FAST binding domains as in [53] and the black CATTTG indicates two putative Mad binding sites. (B) Chromatin isolated from embryos exposed to NaB from stage 1-7 and allowed to develop until stage 21 in 0.1X MMR was used for ChIP with anti-acetyl H4, anti-acetyl H3, anti-H3K4me2 and rabbit IgG (Control) followed by qPCR analysis against the promoter region (gray bar) and intronic region (black bar) of XNr-1. The levels of acetylated H4 and H3 and H3K4me2 were increased only on the intronic region (black bars).
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Figure 6. Mad3's biological activity depends on 5HT. (A) Sensorgram of serotonin binding to GST-Mad3. Data represent the difference between the sample cell (GST-Mad3) and the reference cell (GST). A blank run (buffer only) has been subtracted from all curves. Four different analyte (5HT) concentrations (2, 1.5, 1, 0.8 and 0.4 mM were injected). Injection start and stop are indicated by arrows. The data shown are representative of 3 independent experiments. (B) Embryos were injected at stage 1 with Mad3 WT or Mad3-5mut and scored for left-right phenotype at stage 45. Embryos injected with Mad3 WT presented significant levels of heterotaxia (17%, n = 52, p = 0.001) whereas Mad3-5mut injected embryos presented only 2% of heterotaxia (n = 104, p = 0.7) when compared to control group. (C) Co-Immunoprecipitation assay. Xenopus embryos at the 1 cell stage were injected with Mad3 WT-flag (lanes 1,2,3) or Mad3-5mut-flag (lanes 4,5,6) and collected at the stage 7 (64 cells). The whole protein lysates were incubated with anti-5HT antibody followed by protein-A agarose. Lane 1: 10% Input; Lane 2: whole lysate from Mad3 WT-flag injected embryos plus anti-5HT; Lane 3: negative control (whole lysate from Mad3 WT injected embryos plus rabbit IgG); Lane 4: 10% input; Lane 5: whole lysate from Mad3-5mut-flag injected embryos plus anti-5HTLane 6: negative control (whole lysate from Mad3-5mut-flag plus rabbit IgG).
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