Shinya Matsuda

Dpp/BMP is a morphogen that controls the patterning and growth of the Drosophila wing disc. Although endocytic trafficking has been proposed to influence both extracellular Dpp distribution and signaling, how this process shapes and interprets the Dpp gradient under physiological conditions remains unclear due to limitations in visualizing endogenous Dpp. Here, we generated fluorescent protein-tagged functional dpp alleles that allow simultaneous visualization of extracellular and intracellular Dpp distributions. Using these tools, we found that Dynamin-mediated internalization is required for Dpp signaling activation. Interestingly, in contrast to previous studies, Rab5-mediated early endosomal trafficking is dispensable for Dpp spreading and signaling initiation but is required for signal termination by promoting the downregulation of activated receptors. We show that Dpp signaling is terminated at the multivesicular body (MVB), likely through ESCRT-dependent sorting of activated receptors into intraluminal vesicles (ILVs), rather than via Rab7-mediated lysosomal degradation. Notably, blocking MVB formation expanded the Dpp signaling gradient without altering the extracellular Dpp gradient, thus compromising extracellular Dpp gradient interpretation. Together, our findings reveal that the extracellular Dpp gradient is shaped by Dynamin-dependent internalization and interpreted through the duration of intracellular signaling.

Drosophila male germline stem cells (GSCs) reside at the tip of the testis and surround a cluster of niche cells. Decapentaplegic (Dpp) is one of the well-established ligands and has a major role in maintaining stem cells located in close proximity. However, the existence and the role of the diffusible fraction of Dpp outside of the niche have been unclear. Here, using genetically-encoded nanobodies called Morphotraps, we physically block Dpp diffusion without interfering with niche-stem cell signaling and find that a diffusible fraction of Dpp is required to ensure differentiation of GSC daughter cells, opposite of its role in maintenance of GSC in the niche. Our work provides an example in which a soluble niche ligand induces opposed cellular responses in stem cells versus in differentiating descendants to ensure spatial control of the niche. This may be a common mechanism to regulate tissue homeostasis.

Dpp/BMP acts as a morphogen to provide positional information in the Drosophila wing disc. Key cell-surface molecules to control Dpp morphogen gradient formation and signaling are heparan sulfate proteoglycans (HSPGs). In the wing disc, two HSPGs, the glypicans Division abnormally delayed (Dally) and Dally-like (Dlp) have been suggested to act redundantly to control these processes through direct interaction of their heparan sulfate (HS) chains with Dpp. Based on this assumption, a number of models on how glypicans control Dpp gradient formation and signaling have been proposed, including facilitating or hindering Dpp spreading, stabilizing Dpp on the cell surface, or recycling Dpp. However, how distinct HSPGs act remains largely unknown. Here, we generate genome-engineering platforms for the two glypicans and find that only Dally is critical for Dpp gradient formation and signaling through interaction of its core protein with Dpp. We also find that this interaction is not sufficient and that the HS chains of Dally are essential for these functions largely without interacting with Dpp. We provide evidence that the HS chains of Dally are not essential for spreading or recycling of Dpp but for stabilizing Dpp on the cell surface by antagonizing receptor-mediated Dpp internalization. These results provide new insights into how distinct HSPGs control morphogen gradient formation and signaling during development.

Dpp/BMP acts as a morphogen to provide positional information in the Drosophila wing disc. Key cell-surface molecules to control Dpp morphogen gradient and signaling are heparan sulfate proteoglycans (HSPGs). In the wing discs, two HSPGs, the glypicans Division abnormally delayed (Dally) and Dally-like (Dlp) have been suggested to act redundantly to control these processes through direct interaction of their heparan sulfate (HS) chains with Dpp. Based on this assumption, a number of models on how glypicans control Dpp gradient formation and signaling have been proposed, including facilitating or hindering Dpp spreading, stabilizing Dpp on the cell surface, or recycling Dpp. However, how distinct HSPGs act remains largely unknown. Here we generate genome-engineering platforms for the two HSPGs and find that only Dally is critical for Dpp gradient formation and signaling through interaction of its core protein with Dpp. Nevertheless, we find that this interaction is not sufficient, and that the HS chains of Dally are essential for Dpp gradient formation and signaling largely without interacting with Dpp. We provide evidence that the HS chains of Dally are not essential for spreading or recycling of Dpp but for stabilizing Dpp on the cell surface by antagonizing Dpp internalization through Tkv. These results provide new insights into how distinct HSPGs control morphogen gradient formation and signaling during development.

Secreted signaling molecules act as morphogens to control patterning and growth in many developing tissues. Since locally produced morphogens spread to form a concentration gradient in the surrounding tissue, spreading is generally thought to be the key step in the non‐autonomous actions. Here, we review recent advances in tool development to investigate morphogen function using the role of decapentaplegic (Dpp)/bone morphogenetic protein (BMP)‐type ligand in the Drosophila wing disc as an example. By applying protein binder tools to distinguish between the roles of Dpp spreading and local Dpp signaling, we found that Dpp signaling in the source cells is important for wing patterning and growth but Dpp spreading from this source cells is not as strictly required as previously thought. Given recent studies showing unexpected requirements of long‐range action of different morphogens, manipulating endogenous morphogen gradients by synthetic protein binder tools could shed more light on how morphogens act in developing tissues.

Dpp/BMP is a morphogen that controls patterning and growth in the Drosophila wing disc. Endocytic trafficking has been proposed to control extracellular Dpp spreading or removal, and Dpp signaling. However, how Dpp morphogen gradient is shaped and interpreted by endocytic trafficking remains unclear due to the lack of tools to visualize Dpp distribution at the physiological level. Here we generate functional fluorescent protein tagged dpp alleles to visualize both extracellular and intracellular Dpp distribution. Using these alleles, we found that, while Dynamin-mediated internalization of Dpp is required for Dpp signal activation, Rab5-mediated trafficking is not required for Dpp spreading or signaling as proposed before but for shutting off Dpp signaling through downregulating activated receptors. We provide evidence that Dpp signaling is terminated at Multivesicular body (MVB) likely through sorting activated receptors into intraluminal vesicles (ILVs) rather than Rab7-mediated lysosomal degradation. We further found that blocking MVB formation expanded Dpp signaling gradient without affecting extracellular Dpp gradient, thus compromising extracellular Dpp gradient interpretation. These results indicate that extracellular Dpp gradient is shaped by Dynamin-mediated internalization of Dpp and interpreted by duration of Dpp signaling.

Dpp/BMP acts as a morphogen to provide positional information in the Drosophila wing disc. Key cell-surface molecules to control Dpp morphogen gradient formation and signaling are heparan sulfate proteoglycans (HSPGs). In the wing disc, two HSPGs, the glypicans Division abnormally delayed (Dally) and Dally-like (Dlp) have been suggested to act redundantly to control these processes through direct interaction of their heparan sulfate (HS) chains with Dpp. Based on this assumption, a number of models on how glypicans control Dpp gradient formation and signaling have been proposed, including facilitating or hindering Dpp spreading, stabilizing Dpp on the cell surface, or recycling Dpp. However, how distinct HSPGs act remains largely unknown. Here we generate genome-engineering platforms for the two glypicans and find that only Dally is critical for Dpp gradient formation and signaling through interaction of its core protein with Dpp. We also find that this interaction is not sufficient and that the HS chains of Dally are essential for these functions largely without interacting with Dpp. We provide evidence that the HS chains of Dally are not essential for spreading or recycling of Dpp but for stabilizing Dpp on the cell surface by antagonizing receptor-mediated Dpp internalization. These results provide new insights into how distinct HSPGs control morphogen gradient formation and signaling during development.

Combinatorial signaling is key to instruct context-dependent cell behaviors. During embryonic development, adult homeostasis and disease, Bone Morphogenetic Proteins (BMPs) act as dimers to instruct specific cellular responses. BMP ligands can form both homo- or heterodimers; however, obtaining direct evidence of the endogenous localization and function of each form has proven challenging. Here, we make use of precise genome editing and direct protein manipulation via protein binders to dissect the existence and functional relevance of BMP homo- and heterodimers in the Drosophila wing imaginal disc. This approach revealed in situ the existence of Dpp (BMP2/4)/Gbb (BMP5/6/7/8) heterodimers. We found that Gbb, despite being produced by all the cells of the wing imaginal disc, is only secreted by the cells in which Dpp is expressed. Dpp and Gbb form a gradient of heterodimers whereas neither Dpp nor Gbb homodimers are evident under endogenous physiological conditions. We find that the formation of heterodimers is critical for obtaining optimal signaling and long-range BMP distribution in the developing wing. These results indicate that Dpp/Gbb heterodimers are the active signal required for epithelial patterning and growth.