After some months working in the Münsterberg lab, I successfully established approaches to interfere with microRNA function in vivo, in developing heart of chick embryos, similar to approaches previously published in somites. Briefly, miR function can be inhibited effectively by microinjection of fluorescently tagged and highly specific antagomirs into the early heart tube. Following incubation for 24-72hrs, localization of FI-antagomir can be visualized and the knock down (KD) or double knock down (DKD) of miR expression confirmed by qPCR. This project will examine heart phenotypes resulting from miR DKD. (Panel A)
To determine whether this interaction is also important in cardiac development, we examined BAF60a/b expression after double-knockdown of miR-1 and miR-133 function using targeted microinjection of specific antagomirs. qPCR analysis showed reduced expression of both miRs after DKD, however, we found no significant derepression of the miR-133/miR-1 targets BAF60a and BAF60b. This suggests that interactions of miRs with their respective target genes are highly tissued specific; we hypothesise that negative regulation of the BAF60 variants in the heart requires additional miRs. Overall our observations were consistent with microarray analysis in mice, where the genetic double knock-out of miR-1and miR-133 had no significant effect on BAF60a/b expression.
To assess the efficacy of antagomir-mediated DKD in chick hearts, we examined the expression of genes reported to be affected in the genetic double knock-out. This confirmed a similar profile in chick as was seen in mouse: increased expression of Kcnmb1 and Myocardin, targets for miR-133 and miR-1 respectively, no change for Ccnd2 or Kcnd2, and negative effects on Eya1 and Six1 transcription factors - a secondary consequence of loss of microRNA function, as they are no direct targets of miR-1 or miR-133. Direct targets are expected to be relatively increased, or ‘derepressed’, in absence of miR function. (Panel B)
To validate multiple predicted miR:target gene interactions I used an established pipeline and perform luciferase assays and this has been complemented by in vivo experiments using antagomirs. Using our DKD approach we will systematically inhibit miRs predicted to target components of the BAF/Brg/Brm complex. It is known that BAF60b is downregulated in cardiomyocytes. However, inhibition of miR-1 did not prevent this negative regulation. We hypothesised that this may be due to redundancy with miR-130, which is highly expressed in the heart and we already confirmed interacts with the 3’UTR of BAF60b (Smarcd2) (unpublished), and moreover, miR-19 can play an important role in this modulation because Baf60a and Baf60b are both predicted targets. Thus, we anticipated that DKD of miR-1/miR-130 will lead to derepression of BAF60b expression. Similarly, BAF60a is a predicted target for miR-101, which we showed by small RNA profiling is enriched in the developing heart. We will perform DKD for miR-133 and miR-101 and examine effects on BAF60a expression. In addition, miR-101 is predicted to target Brg1, which we found was derepressed after inhibition of miR-1. Previous small RNA profiling identified, miR-19, miR-101 and miR-130 as highly enriched during cardiogenesis (unpublished). (Panel C)
Embryos between HH stage 14 and 15 have been used and phenotypes have been examined after 24 hours of incubation, and I observed that the survival rate was smaller on DKO1 embryos in comparison with SCR, whereas in the other two conditions was not significant. Moreover, I analyzed the heart rate in these embryos and I could observe that only DKO1 showed a slow heart rate that could imply a bradycardia but the other injected embryos were normal. (Panel D)
