Extended Data Fig. 3: Disease associated AIRIM and AFG2B variants induces stage-specific growth defects of cerebral organoids.
From: A programmed decline in ribosome levels governs human early neurodevelopment
a Bright-field images of SCVI274 (control) and the AIRIMV190G mutant (V190G) organoids at day 30. Note both control and mutant organoids show cortical plate structure. Scale bar, 1 mm. b. Quantification of organoid size in bright-field images at day 30. Control neural tissue is enlarged relative to mutant (V190G); ****P < 0.0001, Unpaired two-sided t-test with Welch’s correction, n = 12 control organoids and 12 mutant organoids from 2 independent batches, P = 7.371387e-05, error bars are SEM. c. Quantification of organoid size in bright-field images at days 2, 5, 7, 10 and15 (R72W). ****P(Day 10) = 2.28472e-11, ****P(Day 15) = 1.99452e-23. Dunettes multiple comparison Unpaired t-test with Welch’s correction (two-sided). n (Day 2) = 23 control EBs 24 mutant organoids from 3 independent batches, n (Day 5) = 23 control organoids 23 mutant organoids from 3 independent batches, n (Day7) = 21 control organoids 22 mutant organoids from 3 independent batches, n (Day 10) = 39 control organoids 37 mutant organoids from 3 independent batches, n (Day 15) = 21 control organoids 23 mutant organoids from 3 independent batches, error bars are SEM. d. Control and AIRIMR72W derived EBs and organoids at days 2, 5, 10, and 15. Scale bar in day 2 and day 5, 130 μm, Scale bar in day 10 and day 15, 320 μm. 3 independent batches were performed. e. Parental control and AFG2B patient derived EBs and organoids at days 5, 10, and 15. Scale bar, 1 mm. Note organoids from patient derived iPS cells exhibit reduced growth and less elongated neuroepithelial buds on day 15. 2 independent batch were performed. f. Representative images showing the proliferation marker phospho-Histone 3 (pH3, grey) and Ki67 (green) signal in control (parent) and mutant (AFG2B patient) day 15 organoids. Scale bar, 50 μm. g. Quantification of pH3 signal of individual neuroepithelial bud at day 15 control (parent) and mutant (AFG2B patient) organoids. Unpaired two-sided t-test with Welch’s correction. n = 10 control and 14 mutant imaged regions from 2 independent batches. P = 0.8875, error bars are SEM. h. Representative images showing the TUNEL signal in control (parent) and mutant (AFG2B patient) day 15 organoids. Note mutant organoids exhibit significantly more TUNEL signal. Scale bar, 50 μm. i. Quantification of TUNEL signal of individual neuroepithelial bud showing mutant organoids exhibit more TUNEL signal. TUNEL counts were normalized to the area of the imaged bud. **P < 0. 01, Unpaired two-sided t-test with Welch’s correction. n = 10 control and 11 mutant imaged regions from 2 independent batches. P = 0.0044, error bars are SEM.
