Fig. 2: Symmetry breaking and pattern formation in elongated cortical assembloids.
a, Experimental procedure for elongated assembloids using mosaic OrEBs containing CAG>tdTOMATO and non-fluorescent CAG > FGF8-expressing cells. Diff, differentiation medium ± vitamin A. b, Representative images of elongated cortical assembloids at 1 div in the PDMS molds (indicated by white arrows in (i); scale bar, 500 µm), at 7 div after removal from the molds and before Matrigel embedding (ii) or after embedding in large Matrigel droplets ((iii); scale bar, 5 mm), at 120 div in the six-well plate ((iv); scale bar, 5 mm). c, Position of the OrEB on elongated cortical assembloids length (as a percentage) at 15, 60 and 120 div. Values are the mean ± s.d. (n = 9 organoids for 15 div, n = 11 for 60 div and 120 div grown from three independent clones). P values for comparisons among time points (one-way ANOVA Tukey’s multiple-comparisons test) are: 15 div versus 60 div, P = 0.9455; 15 div versus 120 div, P = 0.9781; 60 div versus 120 div, P > 0.9999. d, Images of elongated cortical assembloids generated with the SP8 > GFP transgenic line and mosaic OrEBs at 60 div (scale bars, 500 µm). Right, SP8 > GFP intensity per segment (P, M and D) in individual assembloids. Each segmented line represents an individual elongated cortical assembloid (n > 2 from at least 2 clones). P values are the results of one-way ANOVA among segments per condition (0%, P = 0.9045; 1%, P < 0.0001 and 10%, P = 0.9828). e,f, Images of proximal and distal CPNE8 (e) or NR2F1 (f) stainings (scale bars, 50 µm). Bottom, fraction of CPNE8+ (e) or NR2F1+ cells (f) normalized to total (DAPI+) cells in proximal and distal insets of controls (conCAs) and polCAs at 60 div. Whiskers are min to max, boxes represent the 25th to 75th percentiles (Q1 to Q3) and lines indicate the median; CPNE8: n = 20 insets for P and D from 3 conCAs, n = 42 insets for P and n = 39 for D from 3 polCAs; NR2F1: n = 60 insets for P and D from 6 conCAs, n = 40 insets for P and D from 4 polCAs. P values from one-way ANOVA (Tukey’s multiple-comparisons test) are: P = 0.9988 in CPNE8 proximal conCA versus distal conCAs, P = 0.8509 in NR2F1 proximal conCAs versus distal conCAs, P < 0.0001 for other comparisons; NS, not significant. g,g′, Images of 60 div polCA immunostained with tdTomato in red, DAPI in blue and NR2F1 in white (g) or intensity rainbow (g′). Scale bar, 500 µm.
