Figure 1. WDR5 Induces Rx⁺ Neuroectoderm Differentiation via Its RbBP5 Interaction Surface.

(A and B) WDR5 regulates NE proliferation and differentiation in a dose-dependent manner. WT and WDR5^Dox;Wdr5^KO ESCs were maintained in the Dox-containing ESC media. ESCs were resuspended in SFEBq differentiation (EB day 0) in different concentrations of Dox. Cell proliferation at EB day 4 (A) and Rx-GFP positive NE at EB day 5 (B) were determined.

(C and D) Proliferation and NE differentiation in WDR5^Dox; Wdr5^KO ESCs stably transfected with FLAG-tagged WT or different WDR5 mutants. Upon differentiation, Dox was removed (no Dox) or added back 12 h later (2.0 ug/ml, T12h). Cell proliferation was determined at EB day 4 and relative cell proliferation was normalized to the respective group with Dox as 1.0 (D). Percentage of Rx-GFP⁺ NE cells at EB day 5 was determined by flow cytometry (E).

(E) Effects of WDR5-RbBP5 interaction mutants with or without Dox-induced WT WDR5 rescue on NE differentiation were determined by Rax mRNA qRT-PCR (EB day 6).

(F) WT, but not WDR5-RbBP5 interaction mutant WDR5^Q289E, retained capacity to induce Rax-GFP⁺ NE in Wdr5^KO EBs. Representative day 6 EBs were recorded under microscope using bright field or fluorescence channel. Scale bars: 50 μm.

(G) Expression of Dox-inducible WDR5-RbBP5 interaction mutants was not able to induce Rx-GFP⁺ NE differentiation. WDR5^Dex; Wdr5^KO ESCs (Δ26), maintained in dexamethasone (Dex) containing ES media, which enabled Dex-inducible WDR5 expression to maintain ESC self-renewal, were stably transfected with Dox-inducible forms of various HA-tagged WDR5 mutants. Upon differentiation, Dex was removed and Dox was added 12 h later (2.0 ug/ml, T12h). Percentage of Rx-GFP⁺ NE cells at EB day 7 was determined by flow cytometry.

EV: empty vector backbone control. Data in (A)–(F), (G), (H), and (I) represent mean ± SD (n ≥ 3). **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.001.

See also Figures S1 and S2.