XB-IMG-74274
Xenbase Image ID: 74274
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Fig.7. In addition to the premature induction of cardiac field markers resulting from aberrant activation of GATA4 during gastrulation, the misregulation of Notch signaling results in altered levels of GATA4 transcription in gastrula stage embryos. Embryos were injected with one of the following constructs plus a pink lineage marker: GR-Su(H)VP16 (upregulate Notch signaling), GR-Su(H)DBM (downregulate Notch signaling), Esr9-GR (upregulate one Notch-responsive HES gene), or GATA4-GR (upregulate GATA4). Constructs were induced at stage 10 by the addition of dexamethasone to the culture medium, collected approximately 1 h later, and examined for GATA4, nkx2.5, or tbx5 expression via in situ hybridization. The predominant molecular phenotype (increased or decreased expression) is reported as the percentage of embryos with the observed phenotype calculated across three experiments. When Notch signaling was increased following injection and induction of either GR-Su(H)VP16 or Esr9-GR, GATA4 expression was reduced or ablated (71% of injected/induced embryos, n = 77; 74% of injected/induced embryos, n = 53, respectively). Unlike what is observed when Notch signaling is upregulated, when signaling through Notch was suppressed at these stages, GATA4 expression was expanded (76% of injected/induced embryos, n = 110). Interestingly when the temporally inducible GATA4-GR construct was induced at the onset of gastrulation, the expression of both nkx2.5 (83% of embryos, n = 42) and tbx5 (82% of embryos, n = 61) was activated prematurely in late-gastrula embryos. This phenotype is similar to that following suppression of Notch signaling (Fig. 6). For each construct used, injected embryos not exposed to hormone were also examined in order to determine the level of leakiness of the inducible constructs for these experiments. (B–D) Representative images of an uninjected-control embryo or embryos injected with GR-Su(H)VP16 or GR-Su(H)DBM demonstrating the observed GATA4 expression phenotypes. While GATA4 is detected in cells neighboring the dorsal lip of the blastopore (arrows in B), when Notch signaling is upregulated in cells in this region, GATA4 expression is reduced (arrowhead in C). Conversely, when Notch signaling is downregulated GATA4 is robustly expressed by migrating precardiac cells in addition to expression in cells lining the dorsal lip of the blastopore (arrowheads and arrows, respectively, in D). Embryos in panels B–C are oriented ventrally. The embryo in panel D is oriented laterally, with the blastopore facing down. 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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