Shaping BMP morphogen gradients through enzyme-substrate interactions

Bone morphogenetic proteins (BMPs) regulate dorsal/ventral (D/V) patterning across the animal kingdom; however, the biochemical properties of certain pathway components can vary according to species-specific developmental requirements. For example, Tolloid (Tld)-like metalloproteases cleave vertebrate BMP-binding proteins called Chordins constitutively, while the Drosophila Chordin ortholog, Short gastrulation (Sog), is only cleaved efficiently when bound to BMPs. We identified Sog characteristics responsible for making its cleavage dependent on BMP binding. Chordin-like variants that are processed independently of BMPs changed the steep BMP gradient found in Drosophila embryos to a shallower profile, analogous to that observed in some vertebrate embryos. This change ultimately affected cell fate allocation and tissue size and resulted in increased variability of patterning. Thus, the acquisition of BMP-dependent Sog processing during evolution appears to facilitate long-range ligand diffusion and formation of a robust morphogen gradient, enabling the bistable BMP signaling outputs required for early Drosophila patterning.     

Shaping morphogen gradients

Phosphatidylinositol 3-phosphate directs the endosomal localization of regulatory proteins by binding to FYVE and PX domains. New structures of these domains complexed with the phosphoinositide headgroup show how interactions with phosphate and hydroxyl groups differentiate this lipid from all others.     

Dynamics of maternal morphogen gradients in Drosophila

The first direct studies of morphogen gradients were done in the end of 1980s, in the early Drosophila embryo, which is patterned under the action of four maternally determined morphogens. Since the early studies of maternal morphogens were done with fixed embryos, they were viewed as relatively static signals. Several recent studies analyze dynamics of the anterior, dorsoventral, and terminal patterning signals. The results of these quantitative studies provide critical tests of classical models and reveal new modes of morphogen regulation and readout in one of the most extensively studied patterning systems.     

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.     

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.     

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 ability 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 imperative to the two central themes in gradient formation: active transport facilitating long-range signaling 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.     

Regulation of BMP activity and range in Drosophila wing development

Bone morphogenetic protein (BMP) signaling controls development and maintenance of many tissues. Genetic and quantitative approaches in Drosophila reveal that ligand isoforms show distinct function in wing development. Spatiotemporal control of BMP patterning depends on a network of extracellular proteins Pent, Ltl and Dally that regulate BMP signaling strength and morphogen range. BMP-mediated feedback regulation of Pent, Ltl, and Dally expression provides a system where cells actively respond to, and modify, the extracellular morphogen landscape to form a gradient that exhibits remarkable properties, including proportional scaling of BMP patterning with tissue size and the modulation of uniform tissue growth. This system provides valuable insights into mechanisms that mitigate the influence of variability to regulate cell-cell interactions and maintain organ function.     

Creation of a Sog morphogen gradient in the Drosophila embryo.

A variety of genetic evidence suggests that a gradient of Decapentaplegic (Dpp) activity determines distinct cell fates in the dorsal region of the Drosophila embryo, and that this gradient may be generated indirectly by an inverse gradient of the BMP antagonist Short gastrulation (Sog). It has been proposed that Sog diffuses dorsally from the lateral neuroectoderm where it is produced, and is cleaved and degraded dorsally by the metalloprotease Tolloid (Tld). Here we show directly that Sog is distributed in a graded fashion in dorsal cells and that Tld degradation limits the levels of Sog dorsally. In addition, we find that Dynamin-dependent retrieval of Sog acts in parallel with degradation by Tld as a dorsal sink for active Sog.     

miRNAs and morphogen gradients

Morphogens induce biological diversity by operating in a dose-dependent manner. Here we review recent evidences indicating that microRNAs (miRNAs) are ideally suited to serve the morphogen cause. miRNAs regulate the establishment of morphogen gradients, including TGFβ, Wnt and other growth factors by acting on their secretion, distribution and clearance. miRNA are also critical in receiving cells, establishing context-dependency and threshold responses. Moreover, miRNAs contributes to gene networks that transform the graded activity of a morphogen into robust cell fate decisions. Finally, we discuss in the perspective section the implication of the new ceRNA hypothesis for morphogen biology.     

The hedgehog morphogen and gradients of cell affinity in the abdomen of Drosophila

The adult abdomen of Drosophila is a chain of anterior (A) and posterior (P) compartments. The engrailed gene is active in all P compartments and selects the P state. Hedgehog enters each A compartment across both its anterior and posterior edges; within A its concentration confers positional information. The A compartments are subdivided into an anterior and a posterior domain that each make different cell types in response to Hedgehog. We have studied the relationship between Hedgehog, engrailed and cell affinity. We made twin clones and measured the shape, size and displacement of the experimental clone, relative to its control twin. We varied the perceived level of Hedgehog in the experimental clone and find that, if this level is different from the surround, the clone fails to grow normally, rounds up and sometimes sorts out completely, becoming separated from the epithelium. Also, clones are displaced towards cells that are more like themselves: for example groups of cells in the middle of the A compartment that are persuaded to differentiate as if they were at the posterior limit of A, move posteriorly. Similarly, clones in the anterior domain of the A compartment that are forced to differentiate as if they were at the anterior limit of A, move anteriorly. Quantitation of these measures and the direction of displacement indicate that there is a U-shaped gradient of affinity in the A compartment that correlates with the U-shaped landscape of Hedgehog concentration. Since affinity changes are autonomous to the clone we believe that, normally, each cell's affinity is a direct response to Hedgehog. By removing engrailed in clones we show that A and P cells also differ in affinity from each other, in a manner that appears independent of Hedgehog. Within the P compartment we found some evidence for a U-shaped gradient of affinity, but this cannot be due to Hedgehog which does not act in the P compartment.     

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.     

Formation of the BMP activity gradient in the Drosophila embryo

The dorsoventral axis of the Drosophila embryo is patterned by a gradient of bone morphogenetic protein (BMP) ligands. In a process requiring at least three additional extracellular proteins, a broad domain of weak signaling forms and then abruptly sharpens into a narrow dorsal midline peak. Using experimental and computational approaches, we investigate how the interactions of a multiprotein network create the unusual shape and dynamics of formation of this gradient. Starting from observations suggesting that receptor-mediated BMP degradation is an important driving force in gradient dynamics, we develop a general model that is capable of capturing both subtle aspects of gradient behavior and a level of robustness that agrees with in vivo results.     

Gradients and thresholds: BMP response gradients unveiled in Drosophila embryos

Bone morphogenetic proteins (BMP) direct dorsal–ventral patterning in both invertebrate and vertebrate embryos, with strong evolutionary conservation of molecular components of the pathway. Dorsal–ventral patterning of the early Drosophila embryo is a powerful experimental system to probe mechanisms of BMP gradient formation and interpretation. Recent studies have found that spatial patterns of activated BMP signal transducers in Drosophila go through an unexpected transition: a shallow gradient of weak responses at mid-cellularization changes to a step gradient of stronger responses in cellularized embryos. The transition between two gradients of different shape yields new insights into the progression of Drosophila dorsal–ventral patterning and raises new issues about the mechanisms of gradient formation.     

Computational analysis of BMP gradients in dorsal-ventral patterning of the zebrafish embryo

The genetic network controlling early dorsal-ventral (DV) patterning has been extensively studied and modeled in the fruit fly Drosophila. This patterning is driven by signals coming from bone morphogenetic proteins (BMPs), and regulated by interactions of BMPs with secreted factors such as the antagonist short gastrulation (Sog). Experimental studies suggest that the DV patterning of vertebrates is controlled by a similar network of BMPs and antagonists (such as Chordin, a homologue of Sog), but differences exist in how the two systems are organized, and a quantitative comparison of pattern formation in them has not been made. Here, we develop a computational model in three dimensions of the zebrafish embryo and use it to study molecular interactions in the formation of BMP morphogen gradients in early DV patterning. Simulation results are presented on the dynamics BMP gradient formation, the cooperative action of two feedback loops from BMP signaling to BMP and Chordin synthesis, and pattern sensitivity with respect to BMP and Chordin dosage. Computational analysis shows that, unlike the case in Drosophila, synergy of the two feedback loops in the zygotic control of BMP and Chordin expression, along with early initiation of localized Chordin expression, is critical for establishment and maintenance of a stable and appropriate BMP gradient in the zebrafish embryo.     

Early development of leg and wing primordia in the Drosophila embryo

The development of the leg and wing primordia in the Drosophila embryo has been traced using molecular markers. Distal-less and disconnected gene expression provide molecular labels for the leg primordia throughout embryonic development, disconnected expression in the developing leg primordia depends on Distal-less activity. The leg primordia arise as discrete clusters of cells that occupy well defined positions in the embryonic ectoderm. At later stages of embryogenesis the primordia become morphologically recognizable and are intimately associated with the development of the Keilin's organs. The presumptive leg disc and the Keilin's organ appear to derive from a common primordium. Similarly the Abnormal leg pattern gene provides a molecular label for the wing and haltere primordia. The dorsal thoracic primordia appear to be of independent origin from the legs.     

Planar cell polarity and tissue design: Shaping the Drosophila wing membrane

Planar cell polarity (PCP) describes the orientation of a cell within the plane of an epithelial cell layer. During tissue development, epithelial cells normally align their PCP so that they face in the same direction. This alignment allows cells to move in a common direction, or to generate structures with a common orientation. A classic system for studying the coordination of epithelial PCP is the developing Drosophila wing. The alignment of epithelial PCP during pupal wing development allows the production of an array of cell hairs that point towards the wing tip. Multiple studies have established that the Frizzled (Fz) PCP signaling pathway coordinates wing PCP. Recently, we have found that the same pathway also controls the formation of ridges on the Drosophila wing membrane. However, in contrast to hair polarity, ridge orientation differs between the anterior and posterior wing. How can the Fz PCP pathway generate a different relationship between hair and ridge orientation in different parts of the wing? In this Extra View article, we discuss membrane ridge development drawing upon our recent PLoS Genetics paper and other, published and unpublished, data. We also speculate upon how our findings impact the ongoing debate concerning the interaction of the Fz PCP and Fat/Dachsous pathways in the control of PCP.     

Distinguishing graded and ultrasensitive signalling cascade kinetics by the shape of morphogen gradients in Drosophila

Recently, signalling gradients in cascades of two-state reaction-diffusion systems were described as a model for understanding key biochemical mechanisms that underlie development and differentiation processes in the Drosophila embryo. Diffusion-trapping at the exterior of the cell membrane triggers the mitogen-activated protein kinase (MAPK) cascade to relay an appropriate signal from the membrane to the inner part of the cytosol, whereupon another diffusion-trapping mechanism involving the nucleus reads out this signal to trigger appropriate changes in gene expression. Proposed mathematical models exhibit equilibrium distributions consistent with experimental measurements of key spatial gradients in these processes. A significant property of the formulation is that the signal is assumed to be relayed from one system to the next in a linear fashion. However, the MAPK cascade often exhibits nonlinear dose-response properties and the final remark of Berezhkovskii et al. (2009) is that this assumption remains an important property to be tested experimentally, perhaps via a new quantitative assay across multiple genetic backgrounds. In anticipation of the need to be able to sensibly interpret data from such experiments, here we provide a complementary analysis that recovers existing formulae as a special case but is also capable of handling nonlinear functional forms. Predictions of linear and nonlinear signal relays and, in particular, graded and ultrasensitive MAPK kinetics, are compared.