Dpp acts as a long-range morphogen, which spreads along the A-P axis of the wing primordium to form a signaling gradient. Here we studied how receptor mutant clones affect the Dpp gradient in different transport models, and compared theoretical calculations with experimental data.
One outcome of the modeling was the prediction that RMT and RED mechanisms could be discriminated by analyzing Dpp levels behind receptor mutant clones. While in the transcytosis model these levels should be significantly decreased, they would be almost unaltered in the diffusion model. This difference stems from the uptake of Dpp by its receptors, which is an essential feature for morphogen transport by RMT, but not by RED. Our experimental results revealed that neither GFP:Dpp levels nor Dpp signaling activity is reduced behind receptor mutant clones, excluding a significant role for receptor-mediated transcytosis in Dpp gradient formation. Important support for this conclusion was provided by situations where “islands” of wild-type cells received Dpp signal despite being surrounded by mutant tissue, ruling out the possibility that Dpp reaches the distal side of receptor mutant clones by being transported around the clones. When analyzing the GFP:Dpp distribution in mosaic tissues, we also found that the Dpp levels are not significantly reduced within receptor mutant clones. While this outcome further argues against the RMT model, it is consistent with the “external-unbound limit case scenario,” representing RED with the majority of Dpp not being bound to Tkv. Indeed, in the GOF experiments the ratio of unbound Dpp could be narrowed down to approximately 60%–80%.
If transcytosis is modeled in a receptor-independent
manner (as shown in Text S1
), the effects on Dpp distribution by receptor mutant clones do not differ significantly from those in the restricted extracellular diffusion scenario. Thus, receptor-independent transcytosis, for example via fluid phase uptake, remains a possible mechanism for Dpp gradient formation. Several other studies, however, support the restricted extracellular diffusion model. Based on theoretical grounds, Lander et al. (2002) 
proposed that diffusive mechanisms for Dpp gradient formation are more likely than non-diffusive ones. Moreover, experimental studies on heparan sulfate proteoglycans (HSPGs), in particular glypicans, demonstrated the necessity of an intact ECM for morphogen movement 
. In the Drosophila wing disc, clones mutant for the glypicans Dally and Dally-like (Dlp) disrupted the formation of the Dpp gradient 
. Dally was also shown to bind Dpp 
, to stabilize it on the cell surface 
, and to influence its mobility 
However, although the evidence that glypicans assist extracellular diffusion of Dpp seems compelling, alternative or additional functions of glypicans in Dpp distribution cannot be excluded. For example, a recent study 
suggests that apically localized Dlp binds to the Wingless (Wg) morphogen in the Wg producing region, undergoes internalization, and thereby redistributes Wg to the basolateral compartment where Wg spreads to form a long-range gradient. It is possible that recycling of glypicans is also involved in Dpp relocalization and that this process is important for Dpp movement. Consistent with such a notion, Kicheva et al. (2007) 
reported that dynamin-dependent endocytosis is necessary for Dpp movement. Blocking such a ubiquitous cellular machinery, however, not only inhibits the recycling of receptors and glypicans, but may also change the composition and distribution of glypicans in the ECM, which in turn might impede extracellular diffusion. Given that the phenotypes of our receptor clones fully conform to the simplest model of Dpp movement along the ECM (restricted extracellular diffusion), we favor the view that the main function of glypicans for Dpp gradient formation is to facilitate Dpp diffusion along the ECM.
Our observation that receptor mutant clones do not have a major effect on the Dpp gradient contradicts previous observations by Entchev et al. (2000) 
. In their study, ablation of tkv
in small lateral clones leads to an accumulation of Dpp at the side of the clone facing the source, arguing for a block of Dpp movement within such clones 
. The different results could be explained by the presence of brk
in their genetic setup. The ectopic up-regulation of brk
mutant clones, which in most cases leads to clone elimination 
, most likely also causes drastic changes in the transcriptional program in “escaper” cells. Thus the sharp increase in GFP:Dpp levels at the proximal edge inside tkv
mutant clones (facing the Dpp source) could be accounted for by increased levels of Dpp binding proteins, a theory which is supported by the fact that Dpp accumulation was strictly clone-autonomous and not in cells ahead of the clones 
. In our experimental setup, we avoided such secondary effects by simultaneously removing tkv
together with brk
. As our negative control (Mad brk
clones) shows, the signaling state of these cells (Dpp signaling off, no Brk) does not significantly alter the Dpp profile.
Transport along cytonemes is another proposed model for the dispersal of Dpp (Ramirez-Weber and Kornberg, 1999) 
. In its simplest implementation, this model assumes that imaginal disc cells form filopodial extensions towards the Dpp producing region and that Dpp is shuttled along these extensions by binding to Tkv 
. In this scenario, Tkv GOF clones would not only lead to an increase of receptors inside the clones, but also along the cytonemes, and thus affect the Dpp profile also ahead of the clones. This, however, was not observed in our experiments ( and Figure S4
), and we therefore favor the restricted extracellular diffusion model over the cytoneme model for Dpp gradient formation.
During development morphogens function as short-range or long-range signals in order to specify cell fates within a tissue. For example, during wing disc development the range of Hh signaling is relatively short compared to that of Dpp, with a functional range of approximately 10 cells versus 40 cells, respectively 
. It is likely that properties of the transport system are important determinants of the range of a morphogen. In the restricted diffusion model, morphogen spreading is impeded by ECM proteins and cell surface receptors, which efficiently trap their ligand at the cell surface and direct it to degradation. Thus one mechanism to control the range of a morphogen gradient is regulating the receptor levels 
. Indeed, the Hh as well as the Dpp system appear to make use of this strategy to regulate their range. The Hh signal limits its range by upregulating the expression of its binding receptor Patched (Ptc), while the Dpp signal broadens its range by downregulating the expression of its receptor Tkv 
. The effects of our Tkv LOF and GOF clones on the Dpp profile suggest that the majority of Dpp is not bound to the receptor Tkv. It is tempting to speculate that the Dpp-Tkv binding properties represent an additional property of the Dpp signaling system that facilitates the formation of a long-range gradient, by assuring that the majority of Dpp remains in a free and unbound state. Just like lower receptor levels, a lower binding constant would contribute to the spread of Dpp, due to reduced immobilization and degradation of Dpp. It remains to be seen if the ratio of bound to unbound ligand differs for long- versus short-range morphogens and if this ratio represents a general means to regulate the range of morphogen gradients.