Journal: Cell reports
Article Title: Live Imaging Reveals that the First Division of Differentiating Human Embryonic Stem Cells Often Yields Asymmetric Fates.
doi: 10.1016/j.celrep.2017.09.044
Figure Lengend Snippet: Figure 2. Innate Resistance to WNT Signaling in One Daughter Cell Leads to Asymmetric Primitive Streak Specification in Daughter Cell Pairs (A) Overview of the WNT pathway. (B) Stills taken from live imaging of hESCs differentiating toward primitive streak (top); staining of fixed cells reveals asymmetric LEF1 protein expression (bottom). Scale bar represents 10 mm. (C) Ratio of LEF1 protein expression in pairs of daughter cells derived from hESCs exposed to primitive streak differentiation media (ACTIVIN, BMP4, FGF2, and WNT3A); the ratio of LEF1 expression is plotted against the number of hours since the mother cell divided prior to the end of live imaging. Pie charts enumerate the overall percentage of asymmetric versus symmetric daughter cell pairs. (D) Live imaging of hESCs carrying a Tcf/Lef-TurboGFP:NLS:d2PEST reporter allele revealed that upon treatment with primitive streak differentiation media (ACTIVIN, BMP4, FGF2, and WNT3A), WNT/b-catenin signaling is often only activated in one cell in a daughter cell pair. Scale bar represents 10 mm. (E) Ratio of Tcf/Lef-TurboGFP fluorescence in pairs of daughter cells, plotted against the number of hours since the mother cell divided prior to the end of live imaging. Pie charts enumerate the overall percentage of asymmetric versus symmetric daughter cell pairs. (F) MIXL1-GFP hESCs differentiated in primitive streak differentiation media (ACTIVIN, BMP4, FGF2) containing the GSK3 inhibitor CHIR99021 (3 mM) still differentiated through asymmetric divisions, as shown by still images from live imaging. Immunostaining of fixed cells revealed that the MIXL1-GFP+ daughter also expressed BRACHYURY protein. Scale bar represents 5 mm. (G and H) Ratio of MIXL1-GFP (G) and BRACHYURY (H) protein expression in pairs of CHIR99021-treated daughter cells, plotted against the number of hours since the mother cell divided prior to the end of live imaging. Pie charts enumerate the overall percentage of asymmetric versus symmetric divisions. Data are representative of three to five independent experiments.
Article Snippet: Atop this minimal primitive streak induction condition, WNT3A protein (made in house from WNT3A-producing L cells), RSPO2 protein (10–20 ng/mL; R&D Systems), and/or the small-molecule WNT agonist CHIR99021 (3 mM; Tocris) were added as indicated to potentiateWNT signaling and to drive primitive streak specification.
Techniques: Imaging, Staining, Expressing, Derivative Assay, Immunostaining
