Extended Data Fig. 4: FGF and WNT signaling pathway expression and activity in human MG embedded axioloids.
a–b, Expression patterns of FGF and WNT signaling pathway associated transcripts in human MG exposed axioloids at 96 h of culture (24 h after embedding into MG) along the pseudotime axis. Effectors and negative regulators of both pathways are included. c–g, HCR staining images and signal quantification of MG embedded axioloids at 96 h and 120 h derived from c, 409B2 and d–g, 201B7 Luc iPSC lines. Shown images are representative of 3 independent experiments. c–e, HCR staining of MESP2 in yellow, FGF8 in cyan and WNT3A in magenta and corresponding quantification along the posterior to anterior axis of the signal intensity normalized to the position of the WNT3A signal peak for d, and e, (96 h: 3 independent experiments, n = 8 and 120 h: 3 independent experiments, n = 7). f, HCR staining of LEF1 in cyan, AXIN2 in magenta and WNT3A in yellow and corresponding quantification (3 independent experiments, n = 9). g, HCR staining of DUSP6 in cyan, SPRY4 in magenta, and FGF8 in yellow and corresponding quantification (3 independent experiments, n = 9). Single channel images shown in c correspond to the merged channel images shown in Fig. 3a. h–o, HybISS based visualization and quantification of the spatial expression of WNT and FGF signaling pathway members along the posterior to anterior axis in human axioloids at 96 h (top n = 3) and 120 h (bottom n = 2) of culture; WNT signaling members shown in h–k and FGF signaling members shown in l–o; corresponding quantification of spatial expression patterns for WNT pathway related transcripts shown in i, and k, and for FGF pathway related transcripts shown in m, and o. p, Immunofluorescence staining of phospho-ERK (pERK) in cyan, beta-Catenin (CTNNB1) in magenta and TBX6 in yellow in 201B7 Luc-derived MG embedded axioloids at 96 h. q, Corresponding quantification of the signal intensity along the posterior to anterior axis (3 independent experiments, n = 8). r, Magnification of the posterior-most region of the axioloid shown in p. Lines in d,e,f,g and q correspond to mean values, error bands represent the 95% confidence interval, of which the top and bottom show the 2.5 and 97.5 percentiles for each data point. Lines in i,k,m and o correspond to mean values; error bands represent the SD for each data point. Scale bar is 200 µm. a.u., arbitrary units; n = number of axioloids.
