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. Author manuscript; available in PMC: 2019 Oct 4.
Published in final edited form as: Cell Stem Cell. 2018 Sep 20;23(4):501–515.e7. doi: 10.1016/j.stem.2018.08.008

Figure 6:

Figure 6:

Sox2 represses the respiratory fate and promotes the dorsal (esophageal) lineage.

(A-F) In situ hybridization for nkx2–1 of control (A,C,E) or Sox2 MO-injected (B,D,F) Xenopus endoderm explants analyzed at stage NF35 treated with Bio (GSK3β inhibitor) and Bio+BMP4. (G) Schematic depicting experimental protocol to generate human dorsal (Noggin) and ventral (BMP) AFG cultures. +SOX2 indicates tet-inducible SOX2, while -SOX2 indicates SOX2 CRISPRi. (H-N) Analysis of day 9 AFG cultures patterned along the dorsal-ventral axis, with or without SOX2 knockdown in the dorsal cultures using Dox-inducible CRISPRi on day 3–9; (H-K) IF staining of cultures for SOX2 and NKX2–1 and quantification in (L). (M-N) qPCR analysis for SOX2 and NKX2–1 in response to these patterning conditions. (O-U) Doxycycline-induced expression of exogenous SOX2 in ventral cultures on day 8 and analysis on day 9. (O-R) IF staining of cultures for NKX2–1 and HA-SOX2; and (S-T) qPCR analysis for SOX2 and NKX2–1 in response to patterning conditions. Scale bar = 50 μm for IF images, and 200 μm for Xenopus explant images. See quantification and statistical analysis section for details.