Morphogen control of wing growth through the Fat signaling pathway

Organ growth is influenced by organ patterning, but the molecular mechanisms that link patterning to growth have remained unclear. We show that the Dpp morphogen gradient in the Drosophila wing influences growth by modulating the activity of the Fat signaling pathway. Dpp signaling regulates the expression and localization of Fat pathway components, and Fat signaling through Dachs is required for the effect of the Dpp gradient on cell proliferation. Juxtaposition of cells that express different levels of the Fat pathway regulators four-jointed and dachsous stimulates expression of Fat/Hippo pathway target genes and cell proliferation, consistent with the hypothesis that the graded expression of these genes contributes to wing growth. Moreover, uniform expression of four-jointed and dachsous in the wing inhibits cell proliferation. These observations identify Fat as a signaling pathway that links the morphogen-mediated establishment of gradients of positional values across developing organs to the regulation of organ growth.     

BMP morphogen gradients in flies

Bone morphogenetic proteins (BMPs) act as morphogens to control patterning and growth in a variety of developing tissues in different species. How BMP morphogen gradients are established and interpreted in the target tissues has been extensively studied in Drosophila melanogaster. In Drosophila, Decapentaplegic (Dpp), a homologue of vertebrate BMP2/4, acts as a morphogen to control dorsal–ventral patterning of the early embryo and anterior–posterior patterning and growth of the wing imaginal disc. Despite intensive efforts over the last twenty years, how the Dpp morphogen gradient in the wing imaginal disc forms remains controversial, while gradient formation in the early embryo is well understood. In this review, we first focus on the current models of Dpp morphogen gradient formation in these two tissues, and then discuss new strategies using genome engineering and nanobodies to tackle open questions.     

Read-Out of Dynamic Morphogen Gradients on Growing Domains

Quantitative data from the Drosophila wing imaginal disc reveals that the amplitude of the Decapentaplegic (Dpp) morphogen gradient increases continuously. It is an open question how cells can determine their relative position within a domain based on a continuously increasing gradient. Here we show that pre-steady state diffusion-based dispersal of morphogens results in a zone within the growing domain where the concentration remains constant over the patterning period. The position of the zone that is predicted based on quantitative data for the Dpp morphogen corresponds to where the Dpp-dependent gene expression boundaries of spalt (sal) and daughters against dpp (dad) emerge. The model also suggests that genes that are scaling and are expressed at lateral positions are either under the control of a different read-out mechanism or under the control of a different morphogen. The patterning mechanism explains the extraordinary robustness that is observed for variations in Dpp production, and offers an explanation for the dual role of Dpp in controlling patterning and growth. Pre-steady-state dynamics are pervasive in morphogen-controlled systems, thus making this a probable general mechanism for the scaled read-out of morphogen gradients in growing developmental systems.     

Dpp signaling activity requires Pentagone to scale with tissue size in the growing Drosophila wing imaginal disc

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.     

The Role of Endocytosis during Morphogenetic Signaling

Morphogens are signaling molecules that are secreted by a localized source and spread in a target tissue where they are involved in the regulation of growth and patterning. Both the activity of morphogenetic signaling and the kinetics of ligand spreading in a tissue depend on endocytosis and intracellular trafficking. Here, we review quantitative approaches to study how large-scale morphogen profiles and signals emerge in a tissue from cellular trafficking processes and endocytic pathways. Starting from the kinetics of endosomal networks, we discuss the role of cellular trafficking and receptor dynamics in the formation of morphogen gradients. These morphogen gradients scale during growth, which implies that overall tissue size influences cellular trafficking kinetics. Finally, we discuss how such morphogen profiles can be used to control tissue growth. We emphasize the role of theory in efforts to bridge between scales.A fundamental challenge in biology is to understand how morphologies and complex patterns form in multicellular systems by the collective organization of many cells. Cells divide and undergo apoptosis, and they communicate via signaling pathways that use molecules as information carriers. In tissues, large-scale patterns of gene expression emerge from the coordinated signaling activity and response of many cells. The establishment of such patterns is often guided by long-range concentration profiles of morphogens. Cell divisions and cell rearrangements must be coordinated over large distances to achieve specific tissue sizes and shapes. To unravel how molecular processes and interactions can eventually be responsible for the formation of structures and patterns in tissues during development, it is important to study processes at different scales and understand how different levels of organization are connected. Such an approach becomes strongest if it involves a combination of quantitative experimental studies with theory.In the present article, we discuss several such approaches on different scales with a particular emphasis on theory. Starting from the kinetic and dynamic properties of endosomal networks inside a cell, we discuss transport processes in a tissue that can be related to kinetic trafficking parameters. Such transport processes are then responsible for the formation of graded morphogen concentration profiles. To permit scalable patterns in tissues of different sizes, it has been suggested that morphogen gradients scale during growth. This can be achieved on the tissue level by feedback systems that are sensitive to tissue size and regulate, for example, morphogen degradation. Finally, morphogen gradients that scale with tissue size can provide a system to robustly organize cell division in a large tissue and generate homogeneous growth. Theory can play an important role to bridge scales and understand how molecular and cellular processes can control pattern formation and tissue growth on larger scales.Morphogens are signaling molecules that are secreted in specific regions of developing tissues and can induce signaling activity far from their source. They typically form graded concentration profiles and therefore endow cells with positional information (cells can obtain information about their position in a tissue). Thus, they can guide cells to differentiate into complex morphological patterns. Morphogens also control cell growth and cell division. Because they control both patterning and growth, they may play a key role to coordinate these two processes. Such coordination is important because the size of morphological patterns must adjust during growth, whereas growth influences such patterns. A well-studied morphogen is Decapentaplegic (Dpp), which controls morphogenesis in the imaginal wing disc of developing Drosophila. Consequently, mutations in Dpp or defects in the trafficking pathways that control its graded concentration profiles and signaling affect the formation and structure of the adult wing.The study of morphogens was traditionally approached from a genetic perspective: Which gene products behave like morphogens? Which mutants affect patterning and growth? The realization that morphogens typically operate by a gradient of concentration raised the question of how morphogen gradients are generated. It became clear that the cellular trafficking of morphogens is a key issue for the generation of morphogen profiles. Morphogens are secreted ligands that bind receptors in the plasma membrane. The secretion of the ligands and the concentrations of receptor, ligand, and receptor/ligand complex at the plasma membrane are governed by their trafficking in the cell by vesicular transport. In particular, it was shown that trafficking through the endocytic pathway has an important impact on the formation of morphogen gradients (reviewed in Gonzalez-Gaitan 2003; see Bökel and Brand 2014). This is, to a large extent, how the cells respond to morphogens and contribute to set their local concentrations. To understand functions of morphogens in a tissue, we need to study how the gradient is formed. This, in turn, requires insights into morphogen trafficking through the endocytic pathway. The problem of morphogen behavior, therefore, becomes a problem spanning several levels of complexity: the organ level, the tissue level, the cell level, the organelle level, and the molecular level. Theoretical approaches motivated by physics combined with quantitative experimental approaches provide an ideal framework to understand how these different levels of complexity are intertwined.Two recent discoveries highlighted such integration. (1) The observation that profiles of the morphogen Dpp scale during growth, which implies that the rate of Dpp degradation mediated by the endocytic pathway of each of the cells in the tissue depends on the size of the overall tissue. This suggests that two levels of complexity are linked because cellular trafficking receives cues about the global tissue size. (2) As a result of the changes of the degradation rate that leads to gradient scaling, cells receive an increasing level of signaling. This, in turn, can be used by the cells to decide when to divide. This regulation again involves two levels of complexity because regulation at the endocytic pathway determines the growth properties of the tissue and, ultimately, its final size.In the following, we discuss quantitative approaches to study cellular signaling processes on different scales. Here, the aim is to understand how patterns on large scales can emerge during development from molecular processes and signaling pathways that involve endocytosis and cellular trafficking. We begin by describing trafficking of ligands in the endocytic pathway. We then consider the situation of a morphogen ligand and its impact in gradient formation. Subsequently, we discuss how gradient scaling might be realized. Finally, we discuss how such scaling processes play an important role in the regulation of morphogenetic growth.     

Antagonistic growth regulation by Dpp and Fat drives uniform cell proliferation

We use the Dpp morphogen gradient in the Drosophila wing disc as a model to address the fundamental question of how a gradient of a growth factor can produce uniform growth. We first show that proper expression and subcellular localization of components in the Fat tumor-suppressor pathway, which have been argued to depend on Dpp activity differences, are not reliant on the Dpp gradient. We next analyzed cell proliferation in discs with uniformly high Dpp or uniformly low Fat signaling activity and found that these pathways regulate growth in?a complementary manner. While the Dpp mediator Brinker inhibits growth in the primordium primarily in the lateral regions, Fat represses growth mostly in the medial region. Together, our results indicate that the activities of both signaling pathways are regulated in a parallel rather than sequential manner and that uniform proliferation is achieved by their complementary action on growth.     

A mechanism for morphogen-controlled domain growth

Many developmental systems are organised via the action of graded distributions of morphogens. In the Drosophila wing disc, for example, recent experimental evidence has shown that graded expression of the morphogen Dpp controls cell proliferation and hence disc growth. Our goal is to explore a simple model for regulation of wing growth via the Dpp gradient: we use a system of reaction-diffusion equations to model the dynamics of Dpp and its receptor Tkv, with advection arising as a result of the flow generated by cell proliferation. We analyse the model both numerically and analytically, showing that uniform domain growth across the disc produces an exponentially growing wing disc.     

Regulation of cell proliferation by a morphogen gradient

One model to explain the relationship between patterning and growth during development posits that growth is regulated by the slope of morphogen gradients. The Decapentaplegic (DPP) morphogen controls growth in the Drosophila wing, but the slope of the DPP activity gradient has not been shown to influence growth. By employing a method for spatial, temporal, and quantitative control over gene expression, we show that the juxtaposition of cells perceiving different levels of DPP signaling is essential for medial-wing-cell proliferation and can be sufficient to promote the proliferation of cells throughout the wing. Either activation or inhibition of the DPP pathway in clones at levels distinct from those in surrounding cells stimulates nonautonomous cell proliferation. Conversely, uniform activation of the DPP pathway inhibits cell proliferation in medial wing cells. Our observations provide a direct demonstration that the slope of a morphogen gradient regulates growth during development.     

brinker and optomotor-blind act coordinately to initiate development of the L5 wing vein primordium in Drosophila

The stereotyped pattern of Drosophila wing veins is determined by the action of two morphogens, Hedgehog (Hh) and Decapentaplegic (Dpp), which act sequentially to organize growth and patterning along the anterior-posterior axis of the wing primordium. An important unresolved question is how positional information established by these morphogen gradients is translated into localized development of morphological structures such as wing veins in precise locations. In the current study, we examine the mechanism by which two broadly expressed Dpp signaling target genes, optomotor-blind (omb) and brinker (brk), collaborate to initiate formation of the fifth longitudinal (L5) wing vein. omb is broadly expressed at the center of the wing disc in a pattern complementary to that of brk, which is expressed in the lateral regions of the disc and represses omb expression. We show that a border between omb and brk expression domains is necessary and sufficient for inducing L5 development in the posterior regions. Mosaic analysis indicates that brk-expressing cells produce a short-range signal that can induce vein formation in adjacent omb-expressing cells. This induction of the L5 primordium is mediated by abrupt, which is expressed in a narrow stripe of cells along the brk/omb border and plays a key role in organizing gene expression in the L5 primordium. Similarly, in the anterior region of the wing, brk helps define the position of the L2 vein in combination with another Dpp target gene, spalt. The similar mechanisms responsible for the induction of L5 and L2 development reveal how boundaries set by dosage-sensitive responses to a long-range morphogen specify distinct vein fates at precise locations.     

Lineage-tracing cells born in different domains along the PD axis of the developing Drosophila leg.

Patterning of the developing limbs by the secreted signaling proteins Wingless, Hedgehog and Dpp takes place while the imaginal discs are growing rapidly. Cells born in regions of high ligand concentration may be displaced through growth to regions of lower ligand concentration. We have used a novel lineage-tagging method to address the reversibility of cell fate specification by morphogen gradients. We find that responses to Hedgehog and Dpp in the wing disc are readily reversible. In the leg, we find that cells readily adopt more distal fates, but do not normally shift from distal to proximal fate. However, they can do so if given a growth advantage. These results indicate that cell fate specification by morphogen gradients remains largely reversible while the imaginal discs grow. In other systems, where growth and patterning are uncoupled, nonreversible specification events or 'ratchet' effects may be of functional significance.     

Dpp and Gbb exhibit different effective ranges in the establishment of the BMP activity gradient critical for Drosophila wing patterning

Morphogen gradients ensure the specification of different cell fates by dividing initially unpatterned cellular fields into distinct domains of gene expression. It is becoming clear that such gradients are not always simple concentration gradients of a single morphogen; however, the underlying mechanism of generating an activity gradient is poorly understood. Our data indicate that the relative contributions of two BMP ligands, Gbb and Dpp, to patterning the wing imaginal disc along its A/P axis, change as a function of distance from the ligand source. Gbb acts over a long distance to establish BMP target gene boundaries and a variety of cell fates throughout the wing disc, while Dpp functions at a shorter range. On its own, Dpp is not sufficient to mediate the low-threshold responses at the end points of the activity gradient, a function that Gbb fulfills. Given that both ligands signal through the Tkv type I receptor to activate the same downstream effector, Mad, the difference in their effective ranges must reflect an inherent difference in the ligands themselves, influencing how they interact with other molecules. The existence of related ligands with different functional ranges may represent a conserved mechanism used in different species to generate robust long range activity gradients.     

Expansion-repression mechanism for scaling the Dpp activation gradient in Drosophila wing imaginal discs

Maintaining a proportionate body plan requires the adjustment or scaling of organ pattern with organ size. Scaling is a general property of developmental systems, yet little is known about its underlying molecular mechanisms. Using theoretical modeling, we examine how the Dpp activation gradient in the Drosophila wing imaginal disc scales with disc size. We predict that scaling is achieved through an expansion-repression mechanism [1] whose mediator is the widely diffusible protein Pentagone (Pent). Central to this mechanism is the repression of pent expression by Dpp signaling, which provides an effective size measurement, and the Pent-dependent expansion of the Dpp gradient, which adjusts the gradient with tissue size. We validate this mechanism experimentally by demonstrating that scaling requires Pent and further, that scaling is abolished when pent is ubiquitously expressed. The expansion-repression circuit can be readily implemented by a variety of molecular interactions, suggesting its general utilization for scaling morphogen gradients during development.     

Pentagone: patrolling BMP morphogen signaling

Orchestration of spatial organization by signaling gradients - morphogen gradients - is a fundamental principle in animal development. Despite their importance in tissue patterning and growth, the exact mechanisms underlying the establishment and maintenance of morphogen gradients are poorly understood. Our recent work on BMP (bone morphogenetic protein) morphogen signaling during wing development identified a novel protein, Pentagone (Pent), as a critical regulator of morphogen activity. In the following, we discuss the properties of Pent and its role as a feed-back loop in morphogen gradient formation.     

A characterization of the effects of Dpp signaling on cell growth and proliferation in the Drosophila wing

Cell proliferation and patterning must be coordinated for the development of properly proportioned organs. If the same molecules were to control both processes, such coordination would be ensured. Here we address this possibility in the Drosophila wing using the Dpp signaling pathway. Previous studies have shown that Dpp forms a gradient along the AP axis that patterns the wing, that Dpp receptors are autonomously required for wing cell proliferation, and that ectopic expression of either Dpp or an activated Dpp receptor, Tkv(Q253D), causes overgrowth. We extend these findings with a detailed analysis of the effects of Dpp signaling on wing cell growth and proliferation. Increasing Dpp signaling by expressing Tkv(Q253D) accelerated wing cell growth and cell cycle progression in a coordinate and cell-autonomous manner. Conversely, autonomously inhibiting Dpp signaling using a pathway specific inhibitor, Dad, or a mutation in tkv, slowed wing cell growth and division, also in a coordinate fashion. Stimulation of cell cycle progression by Tkv(Q253D) was blocked by the cell cycle inhibitor RBF, and required normal activity of the growth effector, PI3K. Among the known Dpp targets, vestigial was the only one tested that was required for Tkv(Q253D)-induced growth. The growth response to altering Dpp signaling varied regionally and temporally in the wing disc, indicating that other patterned factors modify the response.     

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.     

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.     

Establishing positional information through gradient dynamics: A lesson from the Hedgehog signaling pathway

A long standing question in developmental biology is how morphogen gradients establish positional information during development. Although the existence of gradients and their role in developmental patterning is no longer in doubt, the ability of cells to respond to different morphogen concentrations has been controversial. In the Drosophila wing disc, Hedgehog (Hh) forms a concentration gradient along the anterior-posterior axis and establishes at least three different gene expression patterns. In a recent study, we challenged the prevailing idea that Hh establishes positional information in a dose-dependent manner and proposed a model in which dynamics of the gradient, resulting from the Hh gene network architecture, determines pattern formation in the wing disc. In this Extra View, we discuss further the methodology used in this study, highlight differences between this and other models of developmental patterning, and also present some questions that remain to be answered in this system.Key words: Hedgehog, developmental patterning, morphogen, dynamics, mathematical modeling     

Formation and maintenance of morphogen gradients: An essential role for the endomembrane system in Drosophila melanogaster wing development

As early as 1964 it was suggested that simple diffusion of morphogens away from their secretion source did not provide an adequate explanation for the formation and maintenance of morphogen gradients. Involvement of the endosome in morphogen distribution models provides an explanation for the slow, directional movement of morphogens, as well as their abilty to form intracellular and extracellular gradients independent of morphogen production rates. Drosophila melanogaster morphogens Wg and Dpp form stable, steep, long-range gradients that specify the polarity of the wing disc. The process of endocytosis is imparative to the two central themes in gradient formation; active transport facilitating long-range signalling, and degradation of morphogen to sustain gradient shape. This review investigates the endomembrane mediated processes of re-secretion, degradation, and argosome transport of Wg and Dpp in the hope that a better understanding of the endomembrane system will contribute to a more accurate and comprehensive model for morphogen gradient formation and maintenance.