XB-IMG-74270
Xenbase Image ID: 74270
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Fig.3. Co-expression and co-induction of GR-Su(H)VP16 and ER-Su(H)DBM rescues normal gene expression. Embryos were co-injected with GR-Su(H)VP16 and/or ER-Su(H)DBM and either dexamethasone (Dex), 17β-estradiol (E2), or both hormones were added to the culture medium at stage 10, when heart field specification begins. Embryos were examined for nkx2.5 expression via in situ hybridization at tailbud stages. The percents on the graph summarizing these data represent the number of embryos from three experiments observed to contain an aberrant molecular phenotype (increase or decrease) on the injected side of the heart field as compared to normal nkx2.5 expression on the uninjected side of the same embryo (n = 50–100 animals for each treatment group). (A) When GR-Su(H)VP16 is injected alone and its expression is induced with dexamethasone to upregulate Notch signaling, nkx2.5 expression is dramatically decreased (treatments: Dex10 82% of embryos, n = 91; no hormone control 5% of embryos, n = 97). (B) Conversely, when ER-Su(H)DBM is injected alone and its expression is induced by 17β-estradiol to suppress Notch signaling, an increase in nkx2.5 gene expression is observed (treatments: E210 75% of embryos, n = 60; no hormone control 17% of embryos, n = 76). (C) Co-injection of GR-Su(H)VP16 and ER-Su(H)DBM plus the addition of only one of the two hormones, resulted in similar molecular phenotypes to those observed in embryos injected with one of the two Notch constructs (compare to A, B). As before, upregulated Notch signaling decreased nkx2.5 expression in the heart field (63% of embryos, n = 54) and downregulation of Notch expanded nkx2.5 expression (68% of embryos, n = 63). However, when the two co-injected Notch constructs were simultaneously induced via addition of both hormones (Dex10 and E210) to the culture medium, a rescue of the individual mutant phenotypes was observed (nkx2.5 normally expressed in 66% of embryos, n = 80). (D) Representative images of embryos co-injected with GR-Su(H)VP16 and ER-Su(H)DBM and either dexamethasone (Dex), 17β-estradiol (E2), or both hormones were added to the culture medium at stage 10. Ablated nkx2.5 expression in the Dex(10) treatment is restored in the dual-hormone treatment. Arrows indicate injected side of embryos. White line marks midline of ventral views of tailbud stage embryos. Image published in: Miazga CM and McLaughlin KA (2009) Copyright © 2009. Image reproduced with permission of the Publisher, Elsevier B. V.
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