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The activity of the Dpp morphogen adapts to tissue size in the growing Drosophila wing imaginal disc, and Pentagone, an important secreted feedback regulator of the Dpp pathway, is required for this adaptation., The wing of the fruit fly, Drosophila melanogaster, with its simple, two-dimensional structure, is a model organ well suited for a systems biology approach. The wing arises from an epithelial sac referred to as the wing imaginal disc, which undergoes a phase of massive growth and concomitant patterning during larval stages. The Decapentaplegic (Dpp) morphogen plays a central role in wing formation with its ability to co-coordinately regulate patterning and growth. Here, we asked whether the Dpp signaling activity scales, i.e. expands proportionally, with the growing wing imaginal disc. Using new methods for spatial and temporal quantification of Dpp activity and its scaling properties, we found that the Dpp response scales with the size of the growing tissue. Notably, scaling is not perfect at all positions in the field and the scaling of target gene domains is ensured specifically where they define vein positions. We also found that the target gene domains are not defined at constant concentration thresholds of the downstream Dpp activity gradients P-Mad and Brinker. Most interestingly, Pentagone, an important secreted feedback regulator of the pathway, plays a central role in scaling and acts as an expander of the Dpp gradient during disc growth., Author Summary Scaling, the fitting of pattern to size, manifests itself in numerous examples around us. During development, individual body parts scale up to fit the overall body size. Starved animals form smaller adults with proportionally smaller parts, and amphibian embryos can form normally proportioned adults after extreme surgical operations. How scaling is achieved is not well understood. Here, we establish the Drosophila wing imaginal disc, the precursor tissue of the adult wing, as a model to study scaling quantitatively during growth. In this model, we define scaling as the preservation of proportions of gene expression domains with tissue size during disc growth. The Decapentaplegic (Dpp) morphogen is known to play a central role in Drosophila wing formation and co-coordinately regulates growth and patterning. We found that as the disc grows, the Dpp response expands and scales with the tissue size. Interestingly, scaling is not perfect at all positions in the field. The scaling of the target gene domains is best where they have a function; Spalt, for example, scales best at the position in the anterior compartment where it helps to form one of the anterior veins of the wing. Analysis of mutants for pentagone, a transcriptional target of Dpp that encodes a secreted feedback regulator of the pathway, indicates that Pentagone plays a key role in scaling the Dpp gradient activity.

The TGF-β homolog Decapentaplegic (Dpp) acts as a secreted morphogen in the Drosophila wing disc, and spreads through the target tissue in order to form a long range concentration gradient. Despite extensive studies, the mechanism by which the Dpp gradient is formed remains controversial. Two opposing mechanisms have been proposed: receptor-mediated transcytosis (RMT) and restricted extracellular diffusion (RED). In these scenarios the receptor for Dpp plays different roles. In the RMT model it is essential for endocytosis, re-secretion, and thus transport of Dpp, whereas in the RED model it merely modulates Dpp distribution by binding it at the cell surface for internalization and subsequent degradation. Here we analyzed the effect of receptor mutant clones on the Dpp profile in quantitative mathematical models representing transport by either RMT or RED. We then, using novel genetic tools, experimentally monitored the actual Dpp gradient in wing discs containing receptor gain-of-function and loss-of-function clones. Gain-of-function clones reveal that Dpp binds in vivo strongly to the type I receptor Thick veins, but not to the type II receptor Punt. Importantly, results with the loss-of-function clones then refute the RMT model for Dpp gradient formation, while supporting the RED model in which the majority of Dpp is not bound to Thick veins. Together our results show that receptor-mediated transcytosis cannot account for Dpp gradient formation, and support restricted extracellular diffusion as the main mechanism for Dpp dispersal. The properties of this mechanism, in which only a minority of Dpp is receptor-bound, may facilitate long-range distribution., Author Summary Morphogens are signaling molecules that trigger specific responses in cells in a concentration-dependent manner. The formation of morphogen gradients is essential for the patterning of tissues and organs. Decapentaplegic (Dpp) is the Drosophila homolog of the bone morphogenic proteins in vertebrates and forms a morphogen gradient along the anterior-posterior axis of the Drosophila wing imaginal disc, a single-cell layered epithelium. Dpp determines the growth and final size of the wing disc and serves as an ideal model system to study gradient formation. Despite extensive studies the mechanism by which morphogen gradients are established remains controversial. In the case of Dpp two mechanisms have been postulated, namely extracellular diffusion and receptor-mediated transcytosis. In the first model Dpp is suggested to move by diffusion through the extracellular matrix of a tissue, whereas in the latter model Dpp is transported through the cells by receptor-mediated uptake and re-secretion. In this work we combined novel genetic tools with mathematical modeling to discriminate between the two models. Our results suggest that the Dpp gradient forms following the extracellular diffusion mechanism. Moreover, our data suggest that the majority of the extracellular Dpp is free and not bound to its receptor, a property likely to play a role for the long-range gradient formation.