Dynamic Interpretation of Hedgehog Signaling in the Drosophila Wing Disc

Morphogens are classically defined as molecules that control patterning by acting at a distance to regulate gene expression in a concentration-dependent manner. In the Drosophila wing imaginal disc, secreted Hedgehog (Hh) forms an extracellular gradient that organizes patterning along the anterior–posterior axis and specifies at least three different domains of gene expression. Although the prevailing view is that Hh functions in the Drosophila wing disc as a classical morphogen, a direct correspondence between the borders of these patterns and Hh concentration thresholds has not been demonstrated. Here, we provide evidence that the interpretation of Hh signaling depends on the history of exposure to Hh and propose that a single concentration threshold is sufficient to support multiple outputs. Using mathematical modeling, we predict that at steady state, only two domains can be defined in response to Hh, suggesting that the boundaries of two or more gene expression patterns cannot be specified by a static Hh gradient. Computer simulations suggest that a spatial “overshoot” of the Hh gradient occurs, i.e., a transient state in which the Hh profile is expanded compared to the Hh steady-state gradient. Through a temporal examination of Hh target gene expression, we observe that the patterns initially expand anteriorly and then refine, providing in vivo evidence for the overshoot. The Hh gene network architecture suggests this overshoot results from the Hh-dependent up-regulation of the receptor, Patched (Ptc). In fact, when the network structure was altered such that the ptc gene is no longer up-regulated in response to Hh-signaling activation, we found that the patterns of gene expression, which have distinct borders in wild-type discs, now overlap. Our results support a model in which Hh gradient dynamics, resulting from Ptc up-regulation, play an instructional role in the establishment of patterns of gene expression.     

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.     

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.     

Self-enhanced ligand degradation underlies robustness of morphogen gradients

Morphogen gradients provide long-range positional information by extending across a developing field. To ensure reproducible patterning, their profile is invariable despite genetic or environmental fluctuations. Common models assume a morphogen profile that decays exponentially. Here, we show that exponential profiles cannot, at the same time, buffer fluctuations in morphogen production rate and define long-range gradients. To comply with both requirements, morphogens should decay rapidly close to their source but at a significantly slower rate over most of the field. Numerical search revealed two network designs that support robustness to fluctuations in morphogen production rate. In both cases, morphogens enhance their own degradation, leading to a higher degradation rate close to their source. This is achieved through reciprocal interactions between the morphogen and its receptor. The two robust networks are consistent with properties of the Wg and Hh morphogens in the Drosophila wing disc and provide novel insights into their function.     

Robustness of positional specification by the Hedgehog morphogen gradient

Spatial gradients of Hedgehog signalling play a central role in many patterning events during animal development, regulating cell fate determination and tissue growth in a variety of tissues and developmental stages. Experimental evidence suggests that many of the proteins responsible for regulating Hedgehog signalling and transport are themselves targets of Hedgehog signalling, leading to multiple levels of feedback within the system. We use mathematical modelling to analyse how these overlapping feedbacks combine to regulate patterning and potentially enhance robustness in the Drosophila wing imaginal disc. Our results predict that the regulation of Hedgehog transport and stability by glypicans, as well as multiple overlapping feedbacks in the Hedgehog response network, can combine to enhance the robustness of positional specification against variability in Hedgehog levels. We also discuss potential trade-offs between robustness and additional features of the Hedgehog gradient, such as signalling range and size regulation.     

Shaping Morphogen Gradients by Proteoglycans

During development, secreted morphogens such as Wnt, Hedgehog (Hh), and BMP emit from their producing cells in a morphogenetic field, and specify different cell fates in a direct concentration-dependent manner. Understanding how morphogens form their concentration gradients to pattern tissues has been a central issue in developmental biology. Various experimental studies from Drosophila have led to several models to explain the formation of morphogen gradients. Over the past decade, one of the main findings in this field is the characterization of heparan sulfate proteoglycan (HSPG) as an essential regulator for morphogen gradient formation. Genetic and cell biological studies have showed that HSPGs can regulate morphogen activities at various steps including control of morphogen movement, signaling, and intracellular trafficking. Here, we review these data, highlighting recent findings that reveal mechanistic roles of HSPGs in controlling morphogen gradient formation.Embryonic development involves many spatial and temporal patterns of cell and tissue organization. These patterning processes are controlled by gradients of morphogens, the “form-generating substances” (Tabata and Takei 2004; Lander 2007). Secreted morphogen molecules, including members of Wnt, Hedgehog (Hh), and transforming growth factor-β (TGF-β) families, are generated from organizing centers and form concentration gradients to specify distinct cell fates in a concentration-dependent manner. Understanding how morphogen gradients are established during development has been a central question in developmental biology. Over the past decade, studies in both Drosophila and vertebrates have yielded important insights in this field. One of the important findings is the characterization of heparan sulfate proteoglycan (HSPG) as an essential regulator for morphogen gradient formation. In this review, we first discuss various models for morphogen movement. Then, we focus on the functions of HSPGs in morphogen movement, signaling, and trafficking.     

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.     

Patched controls the Hedgehog gradient by endocytosis in a dynamin-dependent manner, but this internalization does not play a major role in signal transduction

The Hedgehog (Hh) morphogenetic gradient controls multiple developmental patterning events in Drosophila and vertebrates. Patched (Ptc), the Hh receptor, restrains both Hh spreading and Hh signaling. We report how endocytosis regulates the concentration and activity of Hh in the wing imaginal disc. Our studies show that Ptc limits the Hh gradient by internalizing Hh through endosomes in a dynamin-dependent manner, and that both Hh and Ptc are targeted to lysosomal degradation. We also found that the ptc(14) mutant does not block Hh spreading, as it has a failure in endocytosis. However, this mutant protein is able to control the expression of Hh target genes as the wild-type protein, indicating that the internalization mediated by Ptc is not required for signal transduction. In addition, we noted that both in this mutant and in those not producing Ptc protein, Hh still occurred in the endocytic vesicles of Hh-receiving cells, suggesting the existence of a second, Ptc-independent, mechanism of Hh internalization.     

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.     

Implications of diffusion and time-varying morphogen gradients for the dynamic positioning and precision of bistable gene expression boundaries

The earliest models for how morphogen gradients guide embryonic patterning failed to account for experimental observations of temporal refinement in gene expression domains. Following theoretical and experimental work in this area, dynamic positional information has emerged as a conceptual framework to discuss how cells process spatiotemporal inputs into downstream patterns. Here, we show that diffusion determines the mathematical means by which bistable gene expression boundaries shift over time, and therefore how cells interpret positional information conferred from morphogen concentration. First, we introduce a metric for assessing reproducibility in boundary placement or precision in systems where gene products do not diffuse, but where morphogen concentrations are permitted to change in time. We show that the dynamics of the gradient affect the sensitivity of the final pattern to variation in initial conditions, with slower gradients reducing the sensitivity. Second, we allow gene products to diffuse and consider gene expression boundaries as propagating wavefronts with velocity modulated by local morphogen concentration. We harness this perspective to approximate a PDE model as an ODE that captures the position of the boundary in time, and demonstrate the approach with a preexisting model for Hunchback patterning in fruit fly embryos. We then propose a design that employs antiparallel morphogen gradients to achieve accurate boundary placement that is robust to scaling. Throughout our work we draw attention to tradeoffs among initial conditions, boundary positioning, and the relative timescales of network and gradient evolution. We conclude by suggesting that mathematical theory should serve to clarify not just our quantitative, but also our intuitive understanding of patterning processes.     

Robust Generation and Decoding of Morphogen Gradients

Morphogen gradients play a key role in multiple differentiation processes. Both the formation of the gradient and its interpretation by the receiving cells need to occur at high precision to ensure reproducible patterning. This need for quantitative precision is challenged by fluctuations in the environmental conditions and by variations in the genetic makeup of the developing embryos. We discuss mechanisms that buffer morphogen profiles against variations in gene dosage. Self-enhanced morphogen degradation and pre-steady-state decoding provide general means for buffering the morphogen profile against fluctuations in morphogen production rate. A more specific “shuttling” mechanism, which establishes a sharp and robust activation profile of a widely expressed morphogen, and enables the adjustment of morphogen profile with embryo size, is also described. Finally, we consider the transformation of the smooth gradient profile into sharp borders of gene expression in the signal-receiving cells. The integration theory and experiments are increasingly used, providing key insights into the system-level functioning of the developmental system.In order for a uniform field of cells to differentiate into a reproducible pattern of organs and tissues, cells need to receive information about their position within the field. During development, positional information is often conveyed by spatial gradients of morphogens (Wolpert 1989). In the presence of such gradients, cells are subject to different levels of morphogen, depending on their positions within the field, and activate, accordingly, one of several gene expression cassettes. The quantitative shape of the morphogen gradient is critical for patterning, with cell-fate boundaries established at specific concentration thresholds. Although these general features of morphogen-based patterning are universal, the range and form of the morphogen profile, and the pattern of induced target genes, vary significantly depending on the tissue setting and the signaling pathways used.The formation of a morphogen gradient is a dynamic process, influenced by the kinetics of morphogen production, diffusion, and degradation. These processes are tightly controlled through intricate networks of positive and negative feedback loops, which shape the gradient and enhance its reproducibility between individual embryos and developmental contexts. In the past three decades, many of the components comprising the morphogen signaling cascades have been identified and sorted into pathways, enabling one to start addressing seminal questions regarding their functionality: How is it that morphogen signaling is reproducible from one embryo to the next, despite fluctuations in the levels of signaling components, temperature differences, variations in size, or unequal distribution of components between daughter cells? Are there underlying mechanisms that assure a reproducible response? Are these mechanisms conserved across species, similar to the signaling pathways they control?In this review, we outline insights we gained by quantitatively analyzing the process of morphogen gradient formation. We focus on mechanisms that buffer morphogen profiles against fluctuations in gene dosage, and describe general means by which such buffering is enhanced. These mechanisms include self-enhanced morphogen degradation and pre-steady-state decoding. In addition, we describe a more specific “shuttling” mechanism that is used to generate a sharp and robust profile of a morphogen activity from a source that is broadly produced. We discuss the implication of the shuttling mechanism for the ability of embryos to adjust their pattern with size. Finally, we consider the transformation of the smooth gradient profile into sharp borders of gene expression in the signal-receiving cells.     

Fast-tracking morphogen diffusion

The readout of morphogen concentrations has been proposed to be an essential mechanism allowing embryos to specify cell identities [Wolpert Trends Genet 12 (1996) 359], but theoretical and experimental results have led to conflicting ideas as to how useful concentration gradients can be established. In particular, it has been pointed out that some models of passive extracellular diffusion exhibit traveling waves of receptor saturation, inadequate for the establishment of positional information. Two alternative (but not mutually exclusive) models are proposed here, which are based on recent experimental results highlighting the roles of extracellular glycoproteins and morphogen oligomerization. In the first model, inspired from the interactions of Dally and Dally-like with Wingless and Decapentaplegic in the third-instar Drosophila wing disc, two morphogen populations are considered: one in a cell-membrane phase, and another one in an extracellular matrix phase, which does not interact with receptors; in the second model, inspired from biochemical studies of Sonic Hedgehog, morphogen oligomers are considered to diffuse freely without interacting with receptors. The existence of a dynamic sub-population of freely diffusing morphogen allows the system to establish a gradient of bound receptor that is suitable for the specification of positional information. Recent experimental results are discussed within the framework of these models, as well as further possible experiments. The role of Notum in the setup of the Wg gradient is also shown to be likely not to involve a gradient in Notum distribution, even though Notum is only expressed close to the source of Wg synthesis.     

Improving the understanding of cytoneme-mediated morphogen gradients by in silico modeling

Morphogen gradients are crucial for the development of organisms. The biochemical properties of many morphogens prevent their extracellular free diffusion, indicating the need of an active mechanism for transport. The involvement of filopodial structures (cytonemes) has been proposed for morphogen signaling. Here, we describe an in silico model based on the main general features of cytoneme-meditated gradient formation and its implementation into Cytomorph, an open software tool. We have tested the spatial and temporal adaptability of our model quantifying Hedgehog (Hh) gradient formation in two Drosophila tissues. Cytomorph is able to reproduce the gradient and explain the different scaling between the two epithelia. After experimental validation, we studied the predicted impact of a range of features such as length, size, density, dynamics and contact behavior of cytonemes on Hh morphogen distribution. Our results illustrate Cytomorph as an adaptive tool to test different morphogen gradients and to generate hypotheses that are difficult to study experimentally.     

Regulation of Organ Growth by Morphogen Gradients

Morphogen gradients play a fundamental role in organ patterning and organ growth. Unlike their role in patterning, their function in regulating the growth and the size of organs is poorly understood. How and why do morphogen gradients exert their mitogenic effects to generate uniform proliferation in developing organs, and by what means can morphogens impinge on the final size of organs? The decapentaplegic (Dpp) gradient in the Drosophila wing imaginal disc has emerged as a suitable and established system to study organ growth. Here, we review models and recent findings that attempt to address how the Dpp morphogen contributes to uniform proliferation of cells, and how it may regulate the final size of wing discs.     

Drosophila glypicans Dally and Dally-like shape the extracellular Wingless morphogen gradient in the wing disc

Drosophila Wingless (Wg) is the founding member of the Wnt family of secreted proteins. During the wing development, Wg acts as a morphogen whose concentration gradient provides positional cues for wing patterning. The molecular mechanism(s) of Wg gradient formation is not fully understood. Here, we systematically analyzed the roles of glypicans Dally and Dally-like protein (Dlp), the Wg receptors Frizzled (Fz) and Fz2, and the Wg co-receptor Arrow (Arr) in Wg gradient formation in the wing disc. We demonstrate that both Dally and Dlp are essential and have different roles in Wg gradient formation. The specificities of Dally and Dlp in Wg gradient formation are at least partially achieved by their distinct expression patterns. To our surprise, although Fz2 was suggested to play an essential role in Wg gradient formation by ectopic expression studies, removal of Fz2 activity does not alter the extracellular Wg gradient. Interestingly, removal of both Fz and Fz2, or Arr causes enhanced extracellular Wg levels, which is mainly resulted from upregulated Dlp levels. We further show that Notum, a negative regulator of Wg signaling, downregulates Wg signaling mainly by modifying Dally. Last, we demonstrate that Wg movement is impeded by cells mutant for both dally and dlp. Together, these new findings suggest that the Wg morphogen gradient in the wing disc is mainly controlled by combined actions of Dally and Dlp. We propose that Wg establishes its concentration gradient by a restricted diffusion mechanism involving Dally and Dlp in the wing disc.     

Tow (Target of Wingless), a novel repressor of the Hedgehog pathway in Drosophila

Hedgehog (Hh) signalling plays a crucial role in the development and patterning of many tissues in both vertebrates and invertebrates. Aberrations in this pathway lead to severe developmental defects and cancer. Hh signal transduction in receiving cells is a well studied phenomenon; however questions still remain concerning the mechanism of repression of the pathway activator Smoothened (Smo) in the absence of Hh. Here we describe a novel repressor of the Hh pathway, Target of Wingless (Tow). Tow represents the Drosophila homolog of a conserved uncharacterised protein family. We show that Tow acts in Hh receiving cells, where its overexpression represses all levels of Hh signalling, and that this repression occurs upstream or at the level of Smo and downstream of the Hh receptor Patched (Ptc). In addition, we find that like Ptc, overexpression of Tow causes an accumulation of lipophorin in the wing disc. We demonstrate that loss of tow enhances different ptc alleles in a similar manner to another pathway repressor, Suppressor of Fused (SuFu), possibly through mediating Ptc dependant lipophorin internalisation. Combined, these results demonstrate that Tow is an important novel regulator of the Hh pathway in the wing imaginal disc, and may shed light on the mechanism of Ptc repression of Smo.     

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.