Formation of the long range Dpp morphogen gradient

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.     

Dally regulates Dpp morphogen gradient formation by stabilizing Dpp on the cell surface

Decapentaplegic (Dpp), a Drosophila homologue of bone morphogenetic proteins, acts as a morphogen to regulate patterning along the anterior-posterior axis of the developing wing. Previous studies showed that Dally, a heparan sulfate proteoglycan, regulates both the distribution of Dpp morphogen and cellular responses to Dpp. However, the molecular mechanism by which Dally affects the Dpp morphogen gradient remains to be elucidated. Here, we characterized activity, stability, and gradient formation of a truncated form of Dpp (Dpp^ΔN), which lacks a short domain at the N-terminus essential for its interaction with Dally. Dpp^ΔN shows the same signaling activity and protein stability as wild-type Dpp in vitro but has a shorter half-life in vivo, suggesting that Dally stabilizes Dpp in the extracellular matrix. Furthermore, genetic interaction experiments revealed that Dally antagonizes the effect of Thickveins (Tkv; a Dpp type I receptor) on Dpp signaling. Given that Tkv can downregulate Dpp signaling by receptor-mediated endocytosis of Dpp, the ability of dally to antagonize tkv suggests that Dally inhibits this process. Based on these observations, we propose a model in which Dally regulates Dpp distribution and signaling by disrupting receptor-mediated internalization and degradation of the Dpp-receptor complex.     

Dally regulates Dpp morphogen gradient formation in the Drosophila wing

Decapentaplegic (Dpp), a Drosophila TGF beta/bone morphogenetic protein homolog, functions as a morphogen to specify cell fate along the anteroposterior axis of the wing. Dpp is a heparin-binding protein and Dpp signal transduction is potentiated by Dally, a cell-surface heparan sulfate proteoglycan, during assembly of several adult tissues. However, the molecular mechanism by which the Dpp morphogen gradient is established and maintained is poorly understood. We show evidence that Dally regulates both cellular responses to Dpp and the distribution of Dpp morphogen in tissues. In the developing wing, dally expression in the wing disc is controlled by the same molecular pathways that regulate expression of thick veins, which encodes a Dpp type I receptor. Elevated levels of Dally increase the sensitivity of cells to Dpp in a cell autonomous fashion. In addition, dally affects the shape of the Dpp ligand gradient as well as its activity gradient. We propose that Dally serves as a co-receptor for Dpp and contributes to shaping the Dpp morphogen gradient.     

HSPG modification by the secreted enzyme Notum shapes the Wingless morphogen gradient

The secreted signaling protein Wingless acts as a morphogen to pattern the imaginal discs of Drosophila. Here we report identification of a secreted repressor of Wingless activity, which we call Notum. Loss of Notum function leads to increased Wingless activity by altering the shape of the Wingless protein gradient. When overexpressed, Notum blocks Wingless activity. Notum encodes a member of the alpha/beta-hydrolase superfamily, with similarity to pectin acetylesterases. We present evidence that Notum influences Wingless protein distribution by modifying the heparan sulfate proteoglycans Dally-like and Dally. High levels of Wingless signaling induce Notum expression. Thus, Wingless contributes to shaping its own gradient by regulating expression of a protein that modifies its interaction with cell surface proteoglycans.     

Fat facets deubiquitylation of Medea/Smad4 modulates interpretation of a Dpp morphogen gradient

The ability of secreted Transforming Growth Factor β (TGFβ) proteins to act as morphogens dictates that their influence be strictly regulated. Here, we report that maternally contributed fat facets (faf; a homolog of USP9X/FAM) is essential for proper interpretation of the zygotic Decapentaplegic (Dpp) morphogen gradient that patterns the embryonic dorsal-ventral axis. The data suggest that the loss of faf reduces the activity of Medea (a homolog of Smad4) below the minimum necessary for adequate Dpp signaling and that this is likely due to excessive ubiquitylation on a specific lysine. This study supports the hypothesis that the control of cellular responsiveness to TGFβ signals at the level of Smad4 ubiquitylation is a conserved mechanism required for proper implementation of a morphogen gradient.     

The Decapentaplegic morphogen gradient: from pattern formation to growth regulation

Morphogens have been linked to numerous developmental processes, including organ patterning and the control of organ size. Here we review how different experimental approaches have led to an unprecedented level of molecular knowledge about the patterning role of the Drosophila melanogaster morphogen Decapentaplegic (DPP, the homologue of vertebrate bone morphogenetic protein, or BMP), the first validated secreted morphogen. In addition, we discuss how little is known about the role of the DPP morphogen in the control of organ growth and organ size. Continued efforts to elucidate the role of DPP in D. melanogaster is likely to shed light on this fundamental question in the near future.     

A morphogen gradient model for pattern regulation. II. Time description of global morphogen formation and field compartmentalization

In a model for pattern regulation, use was made of local and global morphogens S and Sigma. Sigma is produced from the S-degradation and it is decomposed by first order kinetics while it diffuses along the field. We solve exactly the partial differential equation for the distribution of Sigma in one spatial dimension when its source S is monotonie (for simplicity, linear or generally a power function). Assuming that S and Sigma react reversibly with an allosteric protein P according to a sequential scheme, we derive the evolution in time of the field separation into compartments. At equilibrium the relative extent of each compartment is constant (for variable field size) and so pattern regulation is achieved.     

Spatiotemporal mechanisms of morphogen gradient interpretation

Few mechanistic ideas from the pre-molecular era of biology have had as enduring an impact as the morphogen concept. In the classical view, cells in developing embryos obtain positional information by measuring morphogen concentrations and comparing them with fixed concentration thresholds; as a result, graded morphogen distributions map into discrete spatial arrangements of gene expression. Recent studies on Hedgehog and other morphogens suggest that establishing patterns of gene expression may be less a function of absolute morphogen concentrations, than of the dynamics of signal transduction, gene expression, and gradient formation. The data point away from any universal model of morphogen interpretation and suggest that organisms use multiple mechanisms for reading out developmental signals in order to accomplish specific patterning goals.     

Morpheus unbound: reimagining the morphogen gradient

The theory that the spatial organization of cell fate is orchestrated by gradients of diffusing molecules was a major contribution to 20th century developmental biology. Although the existence of morphogens is no longer in doubt, studies on the formation and function of their gradients have yielded far more puzzles than answers. On close inspection, every morphogen gradient seems to use a rich array of regulatory mechanisms, suggesting that the tasks carried out by such systems are far more extensive than previously thought.     

Dpp gradient formation in the Drosophila wing imaginal disc

The secreted signaling protein Dpp acts as a morphogen to pattern the anterior-posterior axis of the Drosophila wing. Dpp activity is required in all cells of the developing wing imaginal disc, but the ligand gradient that supports this activity has not been characterized. Here we make use of a biologically active form of Dpp tagged with GFP to examine the ligand gradient. Dpp-GFP forms an unstable extracellular gradient that spreads rapidly in the wing disc. The activity gradient visualized by MAD phosphorylation differs in shape from the ligand gradient. The pMAD gradient adjusted to compartment size when this was experimentally altered. These observations suggest that the Dpp activity gradient may be shaped at the level of receptor activation.     

Quantifying the Gurken morphogen gradient in Drosophila oogenesis

Quantitative information about the distribution of morphogens is crucial for understanding their effects on cell-fate determination, yet it is difficult to obtain through direct measurements. We have developed a parameter estimation approach for quantifying the spatial distribution of Gurken, a TGFalpha-like EGFR ligand that acts as a morphogen in Drosophila oogenesis. Modeling of Gurken/EGFR system shows that the shape of the Gurken gradient is controlled by a single dimensionless parameter, the Thiele modulus, which reflects the relative importance of ligand diffusion and degradation. By combining the model with genetic alterations of EGFR levels, we have estimated the value of the Thiele modulus in the wild-type egg chamber. This provides a direct characterization of the shape of the Gurken gradient and demonstrates how parameter estimation techniques can be used to quantify morphogen gradients in development.     

Stability and nuclear dynamics of the bicoid morphogen gradient

Patterning in multicellular organisms results from spatial gradients in morphogen concentration, but the dynamics of these gradients remain largely unexplored. We characterize, through in vivo optical imaging, the development and stability of the Bicoid morphogen gradient in Drosophila embryos that express a Bicoid-eGFP fusion protein. The gradient is established rapidly (approximately 1 hr after fertilization), with nuclear Bicoid concentration rising and falling during mitosis. Interphase levels result from a rapid equilibrium between Bicoid uptake and removal. Initial interphase concentration in nuclei in successive cycles is constant (+/-10%), demonstrating a form of gradient stability, but it subsequently decays by approximately 30%. Both direct photobleaching measurements and indirect estimates of Bicoid-eGFP diffusion constants (D < or = 1 microm(2)/s) provide a consistent picture of Bicoid transport on short ( approximately min) time scales but challenge traditional models of long-range gradient formation. A new model is presented emphasizing the possible role of nuclear dynamics in shaping and scaling the gradient.