Fig. 7: AID2-mediated degradation of endogenous RCC1 causes severe chromosome segregation defects in medaka early embryos.

a Schematic representation of auxin-inducible degron 2 (AID2)-mediated RCC1 degradation. b Procedure of AID-mediated protein knockdown in medaka fertilized eggs. c, d Representative live-cell images showing the fluorescence of RCC1-mAID-mClover-3xFLAG (mACF) and mCh-H2B in control (c) and 5-Ph-IAA-treated (d) embryos. e Quantification of fluorescence intensity of RCC1-mACF in the 4-cell-stage nuclei in control and RCC1-knockdown blastomeres (left). Right graphs show that remining RCC1 levels correlate with phenotypic severity. f Quantification of the frequency of abnormal chromosome segregation in control and RCC1-knockdown 4-cell blastomeres (left). Most daughter cells from normally dividing RCC1-depleted 4-cell blastomeres (n = 9, white) show a non-disjunction phenotype in the subsequent 8-cell division (right). g Live-cell images of control (left) and RCC1-depleted (3 columns on the right) 4-cell blastomeres showing chromosome segregation phenotypes: normal (white), lagging chromosomes (yellow), chromosome bridges (red), and chromosome nod-disjunction (purple). Anaphase lagging chromosomes (arrows) result in micronucleus formation. h Phase contrast images 2 days after mRNA injection and treatment with DMSO (top) or 5-Ph-IAA (bottom) showing lethality in an RCC1-depleted embryo (bottom). i Scatter plots of nuclear size in control and RCC1-knockdown embryos. j Graphs showing frequency of symmetrical or asymmetrical nuclear shape at NEBD in RCC1-KD blastomeres which showed a chromosome bridge or chromosome non-disjunction phenotype. k Width and length of metaphase plates in g are plotted as colored circles for individual 4-cell spindles in control (white, n = 12), and metaphase cells with lagging chromosomes (yellow, n = 6) and chromosome bridging (red, n = 14). Error bars indicate mean ± SD. Scale bars = 100 μm. Source data for (e, f, i–k) are provided as a Source Data file. Two-sided Welch’s t-tests were performed for (e). *p < 0.1, **p < 0.01, and ****p < 0.0001.