Free Extracellular Diffusion Creates the Dpp Morphogen Gradient of the Drosophila Wing Disc

Abstract

SUMMARY Background How morphogen gradients form has long been a subject of controversy. The strongest support for the view that morphogens do not simply spread by free diffusion has come from a variety of studies of the Decapentaplegic (Dpp) gradient of the Drosophila larval wing disc. Results In the present study, we initially show how the failure, in such studies, to consider the coupling of transport to receptor-mediated uptake and degradation has led to estimates of transport rates that are orders of magnitude too low, lending unwarranted support to a variety of hypothetical mechanisms, such as “planar transcytosis” and “restricted extracellular diffusion”. Using several independent dynamic methods, we obtain data that are inconsistent with such models, and that show directly that Dpp transport occurs by simple, rapid diffusion in the extracellular space. We discuss the implications of these findings for other morphogen systems in which complex transport mechanisms have been proposed. Conclusions We believe that these findings resolve a major, longstanding question about morphogen gradient formation, and provide a solid framework for interpreting experimental observations of morphogen gradient dynamics.

Dynamic scaling of morphogen gradients on growing domains

“…3g). Thus, much as in measurements [26][27][28] , only about 3% of ligand represents free, extracellular ligand, while at least 85% of Dpp ligand resides inside cells and another 12% is absorbed on cells. Importantly, scaling is observed for all distinct ligand pools, that is, for the extracellular Dpp ligand alone (Supplementary Fig.…”

“…1e) in a way that the gradient scales with domain size (Fig. 1f) [26][27][28] . Cell-bound and internalized ligand cannot but move passively with the cells as the cells are pushed out during tissue growth (Fig.…”

“…2d,e). For advection to be an efficient and functional transport mechanism, a large fraction of the ligand must be either bound to the cell surface or must be found inside cells [26][27][28] , internalized ligand-receptor complexes must remain stable for a long time, must be partitioned about equally during cell divisions 29 and must remain active after cell divisions 29 . All these conditions have previously been shown to apply to Dpp transport in the Drosophila wing imaginal disc 21,23,24,29 and are likely to also apply to other morphogen transport systems.…”

“…Dpp is produced at rate r Dpp in a narrow stripe at the boundary of the anterior (A) and posterior (P) compartments in the Drosophila wing imaginal disc, and the ligands diffuse into the target domain where they interact with their receptors (Tkv) on cells. Experiments demonstrate that most ligand is found inside cells [26][27][28] , and we therefore use a model 2 that also considers receptor binding and unbinding (at rates k on and k off ), and ligand internalization, and thus comprises diffusible ligand, internal and membrane-bound receptors, as well as receptor-ligand complexes (Fig. 3a, Methods).…”

Essential basal cytonemes take up Hedgehog in the Drosophila wing imaginal disc

“…cDNAs into living embryos (Sanders et al, 2013;Toyoda et al, 2010), by injection of in vitro-synthesized mRNAs into embryos (Muller et al, 2012), and from transgenes under heterologous regulation such as the Gal4-UAS system (Callejo et al, 2011;Kicheva et al, 2007;Zhou et al, 2012). Protein dispersing from expressing cells has been detected by fluorescent imaging, but because expression at an ectopic site can have unforeseen consequences if the systems that normally process and release the morphogen are overwhelmed, or if subcellular structures that are involved are influenced by expression levels, even these direct measures can be problematic in contexts of overexpression and abnormal regulation.…”