Patterning and growth control in vivo by an engineered GFP gradient

Abstract

Morphogen gradients provide positional information during development. To uncover the minimal requirements for morphogen gradient formation, we have engineered a synthetic morphogen in Drosophila wing primordia. We show that an inert protein, green fluorescent protein (GFP), can form a detectable diffusion-based gradient in the presence of surface-associated anti-GFP nanobodies, which modulate the gradient by trapping the ligand and limiting leakage from the tissue. We next fused anti-GFP nanobodies to the receptors of Dpp, a natural morphogen, to render them responsive to extracellular GFP. In the presence of these engineered receptors, GFP could replace Dpp to organize patterning and growth in vivo. Concomitant expression of glycosylphosphatidylinositol (GPI)–anchored nonsignaling receptors further improved patterning, to near–wild-type quality. Theoretical arguments suggest that GPI anchorage could be important for these receptors to expand the gradient length scale while at the same time reducing leakage.

Quantitative analyses reveal extracellular dynamics of Wnt ligands in Xenopus embryos

“…Furthermore, binding to cell surface molecules such as HSPGs and membrane-tethered antibody was sufficient for visible distribution for artificial secreted proteins ( Figures 1C and 2B ). These findings are similar to recent demonstrations that secreted GFP can be synthetic morphogens with specific scaffold molecules in the Drosophila wing disc ( Stapornwongkul et al, 2020 ) and in cultured cells ( Toda et al, 2020 ). On the other hand, secreted GFP appears visible in some tissues, such as deep cells in early zebrafish embryos ( Yu et al, 2009 ) and developing zebrafish brain ( Veerapathiran et al, 2020 ).…”

Synthetic multistability in mammalian cells

“…Future work should extend MultiFate into a full-fledged synthetic cell fate control system, in which extrinsic signals can be used to navigate cells sequentially through a series of fate choices, recapitulating cell behaviors associated with normal development. Coupling MultiFate to synthetic signaling systems such as synNotch ( 77 , 78 ), MESA ( 79 ), synthekines ( 80 ), orthoIL-2/2R ( 81 ), engineered GFP ( 82 ) and auxin ( 83 ) should enable flexible and orthogonal control. MultiFate could also allow engineering of multicellular cell therapeutic programs.…”

Novel synthetic biology approaches for developmental systems

“…Although many details of the system are known, and the role of Dpp gradient as a morphogen is established, one outstanding question in the field was whether the system could work by diffusion only, as opposed to another mechanism for passing the signal between cells (e.g., long-range filopodia, secreted vesicles). To address this question Stapornwongkul et al (2020) devised a new way to activate the Dpp pathway with genetically encoded synthetic ligands, which allowed them to prove that cells do not read something special about Dpp, but simply its gradient (Figure 3A). Specifically, the authors generated a new synthetic receptor that recognizes GFP as input and activates the Dpp pathway in response (GFP/Dpp receptor).…”

“…These can be used to study more precisely the functions of existing pathways that they mimic. Examples include synthetic Shh (Li et al, 2018) and synthetic Dpp (Stapornwongkul et al, 2020) pathways. In the example of Shh, Li et al (2018) were able to reconstitute both linear and radial morphogen gradients in vitro using a synthetic version of the Hedgehog pathway in the mouse fibroblast NIH/3T3 cell line.…”