Fig. 6

The Isl1-Ajuba Transcriptional Complex Negatively Regulates Isl1 Expression in an RA-Dependent Mechanism (A) Relative expression levels of cardiac progenitor marker mRNA normalized to GAPDH mRNA in day 5 EBs either nontreated or treated with 0.1 or 0.3 µM RA at day 3. Data are mean ± SD (n = 2). *p < 0.05; **p < 0.01; ***p < 0.001. (B) Western blot analysis of nuclear extracts from day 5 EBs either nontreated or treated with 0.1 or 0.3 μM RA at day 3.(C) ChIP of nuclear extracts from day 5 EBs either nontreated or treated with 0.1 μM RA at day 3, using anti-Isl1 and anti-Ajuba antibodies or IgG as a control. PCRs were performed using primers flanking a conserved Isl1 binding site in the Isl1 enhancer. (D and E) P19 cells were transiently transfected with 25 ng Isl1-luc reporter construct (Isl1-F; Kang et al., 2009), alone or together with Isl1 (400 ng), Ajuba (900 ng), or with constant amount of Isl1 (400 ng) and increasing amounts of Ajuba expression plasmid (500 and 900 ng). P19 cell were either nontreated (D) or treated with RA (E). Data are mean ± SD (n = 4). p < 0.01; ***p < 0.001. (F) In situ hybridization for the atrial marker atrial myosin heavy chain (Amhc; top panels) and Isl1 (bottom panels) at 48 hpf in control and zebrafish embryos, treated with RA for 1 hr. WT, wild-type. (G) In situ hybridization for Isl1 in control and Ajuba morphant and RA-treated control or Ajuba morphant zebrafish embryos at 48 hpf. See also Figure S7 and Tables S2 and S3.