XB-IMG-150239
Xenbase Image ID: 150239
|
Figure 7. The direct repression of RRAS by miR-449 affects multiciliogenesis and apical actin cap formation in Xenopus.(a,b) Eight-cell stage Xenopus embryos were injected in the epidermis precursor blastomeres with a mixture of synthetic mRNA coding for GFP-CAAX to label the injected cells (in green, a2,6,10,14) and negative control MO (CTR-Neg, a1–4, b1,2), or a morpholino protecting rras against binding by miR-449 (PO-rras, a5–8, b3,4) or a morpholino blocking the translation of rras (MO-ATG-rras, a9–12), or a combination of PO-rras and MO-ATG-rras (a13–16). MCCs are stained with an anti-acetylated tubulin antibody (in magenta, a3,7,11,15 and c2,4), F-actin with phalloidin (red, a1,5,9,13 and b1–4). (b) The impact of Po-rras in MCCs on apical (upper b5) and sub-apical actin (lower b6) was observed with F-actin staining. (c) In independent experiments, we used rGBD-GFP to examine RhoA activity in Xenopus MCCs (c1–4). Eight-cell stage Xenopus embryos were injected in the epidermis precursor blastomeres with a reporter of RhoA activity (rGBD-GFP in green, c1–4) and with negative control MO (CTR-Neg, c1–2) or with PO-rras (c3–4). MCCs are stained in magenta (c2,4). Protecting the rras mRNA from interaction with miR-449 results in defects in actin cap formation (F-Actin, a5 and b3,4) together with a loss of MCCs (Ac. Tub., a7,c4) without affecting apical RhoA activity (rGBD-GFP, c1,3). This phenotype is rescued when the translation of the protected rras mRNA is blocked by coinjection of MO-ATG-rras (a13–16). (d) The histogram indicates the percentage of injected cells (positive for mGFP) that develop proper apical actin cap (in red) and motile cilia (in magenta) in Xenopus epidermis at stage 25. CTR-Neg, n=10 embryos per 413 injected cells; PO-rras, n=8 embryos per 350 injected cells; MO-ATG-rras, n=8 embryos per 290 injected cells; MO-PO-rras+MO-ATG-rras n=9 embryos per 395 injected cells (***P=0.009 and P<0.0001, and **P=0.0016; Mann–Whitney test). Data are mean±s.e.m. (e) The histogram indicates the percentage of rGBD-GFP-positive cells stained for α-tubulin (α-tub. in black) or acetylated tubulin (Ac. Tub. in magenta) in PO-rras morphants (PO-rras, n=150) in comparison with the negative control (CTR-Neg, n=110). (**P<0.005; ns, no significant; one-way-analysis of variance with Dunnett's test). Data are mean±s.e.m. Image published in: Chevalier B et al. (2015) Copyright © 2015, Nature Publishing Group, a division of Macmillan Publishers Limited. 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 |