Figure 7.

Inherent Tendency to Ventralization Is Largely Rescued by a Brief, Specific Phase of Wnt Signaling Activation

(A) Diagram of Hedgehog pathway components targeted by small molecules in (B) and (C).

(B) Treatment with purmorphamine (1 μM) between 7 and 17 dpi results in a more ventralized gene expression profile at 33 dpi (line iPSC22.1, n = 2).

(C) Treatment with Hedgehog inhibitor cyclopamine (1 μM) between 7 and 17 dpi has no observable effect on dorso-ventral gene expression in a highly ventralized line at ∼35 dpi (iPSC14.1, n = 3).

(D) Summary diagram of Wnt pathway components targeted by small molecules in (E)–(G).

(E) Treatment with Wnt inhibitor IWP2 (2 μM) between 0 and 12 dpi results in a more ventralized gene expression profile at 33 dpi (line iPSC22.1, n = 2).

(F) Treatment with Wnt activator CHIR99021 (1 μM) between 13 and 17 dpi significantly increases cortex-associated gene expression and decreases MGE-associated expression at ∼35 dpi in differentiations of 4 ventral-prone lines compared to vehicle treatment (GMESC01.1, iPSC01.1, iPSC06.1, and iPSC22.1) (one-sample Student’s t test, mu = 0, n = 7, FDR-corrected p values: ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001). Top panels show combined trend; bottom panels show breakdown by PSC line.

(G) Clustering of ∼35-dpi differentiations from ventral-prone lines treated between 7 and 17 dpi with either vehicle or 1 μM CHIR99021. Treatment stimulating Wnt/β-catenin signaling results in shift in classificationof ventralized differentiations to more dorsalized clusters.

(H) Model for outcome of differentiation of distinct cell lines.

All error bars represent standard error. See also Figure S7.