XB-IMG-154848
Xenbase Image ID: 154848
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Figure S3: miR-182 MO efficiently knocks down endogenous miR-182 without affecting the
generation of RGCs nor the growth of their axons, and miR-182 mimics rescues miR-182
expression, Related to Figure 3.
(A-E) Effect of the injection of control or miR-182 MOs in both dorsal blastomeres at eight cell stage
embryos, driving the expression of the morpholino in the central nervous system, in Xenopus laevis
embryos at stage 37/38 and 40. (A) Schematic of the experimental protocol and representative images
of control and miR-182-injected embryos at stage 40. No major difference was observed between
controls and miR-182 morphants morphology. (B) Representative images of ISH signal for control or
miR-182 probes on stage 37/38 control or miR-182 morphant embryos. Most of the ISH signal for
miR-182 in neural cells disappears in presence of miR-182 MOs, suggesting that miR-182 MOs
efficiently knockdown endogenous miR-182. (C) Quantification of the size of the eye of control and
miR-182 morphant embryos at stage 40 shows no significant differences between the two populations.
(D) Representative images of cryosections of the eye of control or miR-182 morphant embryos at stage
40, at the level of the optic nerve head, after immunolabeling for Sox2 (red) and Islet1 (green) and
counterstaining with DAPI (blue). (E) Quantification of the number of RGCs, per section, in stage 40
control or miR-182 morphant embryos. No significant difference was observed. (F) Schematic
representation of the quantification method after injection in dorsal blastomeres or electroporation in
the eye of control or miR-182 MOs, and DiI labeling of the RGC axons. The length of axons was
assessed by measuring the distance from the optic chiasm to the longest RGC axons (l) normalized to
the size of the brain from the optic chiasm to the posterior boundary of the tectum (L). (G-H) Graph
showing the quantification of RGC axon length in control and miR-182 MOs injected (G) or
electroporated (H) embryos. No significant difference in RGC axon length was detected in absence of
miR-182. (I) Representative images of ISH for miR-182 on vibratome section of stage 37/38 morphant
embryos after eye electroporation of control- or miR-182 mimics in the eye. Exogenous miR-182
mimics, but not control mimics, were detected in electroporated retinal areas (delineated by a dashed
black line) in a miR-182 morphant background. Through the figure: values are mean ± SEM. Numbers
of eye/embryo (C), retinal sections (E) and brains (G, H) analyzed are indicated within bars. All
samples passed D’Agostino & Pearson Omnibus normality test. Student’s t-test. ns, nonsignificant
Scale bars, 1mm (A), 250µm (B), 100µm (D,I). Image published in: Bellon A et al. (2017) © 2017 The Authors. Creative Commons Attribution license Larger Image Printer Friendly View |