Xenbase Image ID: 130570

Figure 5. miR-9 Regulates the Expression of hairy1 In Vivo(A) Sequence alignment of the predicted miR-9 binding site in HES1 homologs in human, mouse, Xenopus, and zebrafish. Positions that have a single, fully conserved residue are marked with an asterisk. Seed-complementary region is boxed in red.(B) HeLa cells were transfected with WT Xenopus hairy1 (xhairy1_WT), mouse Hes1 (mHes1), or mutant hairy1 (xHairy1_Mut) reporter together with either scrambled (Control) or miR-9 precursors (miR-9). Luciferase expression was normalized and expressed relative to the control levels. Error bars represent SD.(C) Design of target protector morpholino (Hairy1 TP) directed against hairy1 miR-9 binding site. Seed region is boxed in red.(D) Hairy1 TP alleviates the repression of hairy1 luciferase reporter when cotransfected with miR-9 precursors but has no effect on the repression of other miR-9 targets. Error bars represent SD.(E) In situ hybridization for miR-9 (miR-9a-1 transcript) and hairy1 in stage 30 embryos. Shown are whole mounts and transverse sections through the respective brain areas.(F) Double-fluorescent in situ for hairy1 (red) and miR-9a-1 (green) shows mutually exclusive pattern of expression along the AP axis.(G) miR-9 MO and hairy1 TP lead to expansion of the hairy1-positive domain (red arrowheads) along the AP axis, as shown by in situ hybridization.(H) Quantification of the change in hairy1 mRNA expression using qRT-PCR.(I) Hes1 mRNA levels in N1E neuroblastoma cells are downregulated when miR-9 is overexpressed and increased when it is knocked down using LNA inhibitors. In all graphs, data are presented as mean values, and error bars represent SEM. Image published in: Bonev B et al. (2011) © 2011 ELL & Excerpta Medica. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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