Figure 2: miR-31 inhibition results in embryonic lethality.

(a) Zygotes were injected with miR-31 inhibitor or control miR-124 inhibitor. The number of embryos in each developmental stage was recorded every hour for 6 hours post-fertilization (hpf), then again at 24 hpf. p-values were obtained using Cochran-Mantel-Haenszel test. N=212 Control miR-124 inhibitor injected embryos,128 miR-31 inhibited embryos, 3 biological replicates. (b) miR-31 inhibited embryos exhibit chromosomal bridging, lagging chromosomes (arrows), and uncondensed chromosomes that may result in aneuploidy. Embryos were injected with miR-31 LNA inhibitor or control miR-124 LNA inhibitor and immunolabeled for tubulin in green and counterstained DNA with DAPI in blue. Scale bar = 50μm. Blastomeres undergoing mitosis were scored for chromosomal abnormalities as indicated. p-values were obtained using Cochran-Mantel-Haenszel test. N=138 Control miR-124 inhibitor-injected blastomeres, N=139 miR-31 inhibitor-injected embryos. 3 biological replicates (c) miR-31 inhibition results in increased interpolar and astral microtubules. Embryos were injected with miR-31 LNA inhibitor or control miR-124 LNA inhibitor and immunolabeled for tubulin in green and counterstained DNA with DAPI (blue). ImageJ was used to measure the fluorescence intensity of individual blastomeres during anaphase. N=37 control blastomeres, 30 miR-31 inhibited blastomeres. p-values were obtained using Student’s t-test. (d) Control embryos exhibit enriched filamentous actin (F-actin) at the cell cortex and surrounding the chromosomes. miR-31 inhibited embryos exhibit an increase in F-actin, particularly surrounding the chromosomes. Zygotes were injected with miR-31 LNA inhibitor and labeled with Alexa647-phalloidin (magenta) and counterstained with DAPI (blue). ImageJ was used to measure the fluorescence intensity of single blastomeres. N= 49 control blastomeres, 40 miR-31 inhibited blastomeres. p-values were obtained using Student’s t-test.