Fig. 1. Turning arbitrary proteins into synthetic morphogens.

(A) SynNotch receptors detect juxtacrine signals (e.g., membrane-tethered GFP). In the diffusible synNotch system, soluble GFP is produced from a secretor cell, then trapped by anti-GFP anchor protein, and finally presented to anti-GFP synNotch on a receiver cell. (B) Multiple arbitrary proteins with two recognition sites could be converted into synthetic morphogens (see fig. S3 for construction of mCherry-PNE peptide morphogen). Kd, dissociation constant. (C) Testing diffusible GFP synNotch system in L929 mouse fibroblasts. Anchor cell expresses anti-GFP LaG2 anchor protein. Receiver cell expresses anti-GFP LaG17 synNotch (induces mCherry reporter). 1 × 10⁴ GFP-secreting cells, 0.5 × 10⁴ anchor cells, and 0.5 × 10⁴ receiver cells were cultured overnight, and mCherry induction in receiver cells was measured by flow cytometry. (D) Juxtacrine versus diffusible GFP signaling gradient. Left pole has 3 × 10⁴ sender cells, and right body has 1.5 × 10⁴ cells (100% receiver cells for juxtacrine; 50:50 anchor:receiver cells for diffusible GFP; see fig. S2, A and B). Images were taken by incucyte system over 4 days when system reached steady state (movie S1). Individual lines show mCherry intensity every 12 hours. memb., membrane.