Extracellular diffusion creates the Dpp morphogen gradient of the Drosophila wing disc

During development, cells often receive spatial patterning cues from extracellular signaling molecules known as morphogens. Decapentaplegic (Dpp) is a typical morphogen in the Drosophila wing disc. Because Dpp is a secreted protein, a stable gradient could potentially be set up by diffusion through extracellular spaces, coupled with uptake and degradation by cells. However, several studies have suggested that Dpp may be actively transported into and out of cells (known as "planar transcytosis"). Genetic experiments aiming to address this question have produced conflicting interpretations, no doubt in part because it is difficult to draw meaningful inferences about dynamic processes from static data. Recently, Kicheva et al. (Kicheva et al., 2007) used Fluorescence Recovery After Photobleaching (FRAP) to study the formation of Dpp gradients in wing discs expressing a fusion of Dpp to green fluorescent protein (DppGFP). They claimed the transport coefficient is 0.1 microm2/s, consistent with a model of transport by transcytosis.;Here we revisit this question using a complementary technique: flies were generated expressing a fusion of Dpp to Dendra2, a photoactivatable fluorescent protein. When a localized group of cells is subjected to photoactivating light, no movement of photoactivated DppDendra out of them occurs. The same result is obtained on both short and long time scales, and even when steps are taken to reduce the contribution of an immobile pool that is also detected in FRAP experiments. Although such observations are inconsistent with transport by transcytosis, they are consistent with transport through fast extracellular diffusion. With Fluorescence Correlation Spectroscopy (FCS) and Pair Correlation Function (pCF) microscopy, we observed that Dpp molecules in the wing disc move with a diffusivity of ∼20 microm2/s, consistent with extracellular diffusion.;The results presented here argue that a small amount of freely-diffusing extracellular Dpp exists in the Drosophila wing disc, that it alone is sufficient to account for morphogen gradient formation, and that the vast majority of Dpp associated with cells is irreversibly trapped. Although we cannot rule out the existence of alternate transport mechanisms, the data imply that little, if any, of the total flux of Dpp is carried by such means.