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. 2020 Jul 16;133(14):jcs249557. doi: 10.1242/jcs.249557

Fig. 3.

Fig. 3.

Effect of GDNF on organoid differentiation. (A) Schematic of the GDNF–Ret signaling axis that drives formation of the collecting duct system. Relative mRNA fold change between control (n=4 cultures for each group, blue circle) and GDNF pulse (n=4 cultures for each group, red square) organoids for the stem cell marker, Six2 (P=0.2854, two-tailed Student's t-test), and the downstream GDNF effectors Wnt11 and Gata3 (Wnt 11, P=0.0367; Gata3, P=0.0184; two-tailed Student's t-test). Data shown are mean±s.e.m. (B) ReviGO representation of significantly changed (nominal P<0.05) GO terms in GDNF pulsed organoids [n=4 cultures, dot size=log10 frequency (percentage of genes annotated) with that GO term (Supek et al., 2011), dot color=log10 P-value]. (C) Principal cell-specific transcription factors are significantly upregulated in GDNF pulsed tubuloids (n=4 cultures for each group, red square) when compared to controls (n=4 cultures for each group, blue circle). Significant upregulation of Gata3 (P=0.0499), Ehf (P=0.0455), Irf1(P=0.0225), Irf9 (P=0.0132), Stat1 (P=0.0220), Nfia (P=0.0168), Maf (P=0.0239), Nfix (P=0.0171), Glis2 (P=0.0175), and Grhl2 (P=0.0789) determined by one-tailed Student's t-test. Data shown are mean±s.e.m. (D) Schematic experimental paradigm for the GDNF pulse. (E) Morphometry tracking of tubuloids. Representative bright-field images of tracked tubule bifurcation events from 48 h to 72 h post-plating. Scale bar: 100 μm. (F) 3D SIM reconstruction of ZO-1 (red) to demonstrate junctional organization in tubuloid structures. Scale bars: 25 μm. (G) Immunofluorescence images of a differentiated spheroid and tubule positive for collecting duct marker, DBA (red) with basolateral Na+/K+-ATPase (green) and DAPI (blue). Scale bars: 50 μm. (H) Quantification of structure classification in bright-field images taken with 4× objective magnification reveals that the addition of GDNF yields more differentiated structures (P=0.0102; two-tailed Student's t-test) that are tubules (P=0.0091; two-tailed Student's t-test.) when compared to control. Data shown are mean±s.e.m. n=574 structures, three tubuloid preparations and three mice for control and n=526 structures, three tubuloid preparations and three mice for +GDNF. **P≤0.01; *P≤0.05; ns, not significant.