Figure 6
LcnRNA expression and modulation in PE/ST explants Panel A. Schematic representation of the genomic location of annotated lncRNAs within Wt1, Bmp4 and Fgf8 loci in chicken. Panel B. qPCR analyses of the expression of lncRNAs in HH17 embryonic heart and PE/ST, respectively. Observe that Wt1_76127, Bmp4_53170 and Fgf8_57126 display significant increased expression in the PE/ST as compared to the embryonic heart at the same developmental stage (HH17). Panel C. qPCR analyses of Wt1_76127, Bmp4_53170 and Fgf8_57126 in thymosine β4 treated PE/ST explants. Observe that thymosine β4 treatment increases Bmp4_53170 while decreased Wt1_76127 and Fgf8_57126 expression. Panel D. qPCR analyses of Wt1_76127, Bmp4_53170 and Fgf8_57126 in Bmp2, Bmp4, Fgf2 and Fgf8 treated PE/ST explants, respectively. Observe that Bmp signaling decreased while Fgf signaling increased the expression of Wt1_76127, Bmp4_53170, while Fgf8_57126 displays the opposite regulatory modulation by Bmp and Fgf signaling. Observe that miR-195 increases Wt1_76127 while decreases Bmp4_53170 expression. On the other hand miR-23 and miR-27 decreases Wt1_76127 while does not modify the expression of Bmp4_53170. Panel E. qPCR analyses of Wt1_76127, Bmp4_53170 and Fgf8_57126 in miR-23, miR-27, miR-195 and miR-223 treated PE/ST explants, respectively. HH17 PE and HH17 embryonic hearts were collected from >30 embryos and pooled to performed RNA isolation. In all cases, three distinct biological replicates of pooled HH17 PE/ST and HH17 heart samples were subsequently tested by qPCR (panel A). HH17 PE/ST were dissected from >30 embryos, treated with thymosine beta4 (panel C), the correspoding Bmp/Fgf growth factor (panel D), and/or microRNA mimics treatment (panel E), respectively and subsequently pooled to performed RNA isolation. On each case, three distinct biological replicates were subsequently tested by qPCR.
