Smiling Gurken gradient: An expansion of the Gurken gradient

Morphogen gradients provide unique positional information within a tissue. Cells that are sensitive to the concentration of the morphogen integrate this signal and develop an appropriately distinct cell fate. A morphogen gradient is usually generated by a restricted source and shaped by the speed of diffusion and stability of the signaling molecule. In addition, the availability of receptor and Heparan Sulfate Proteoglycans (HSPGs) help to shape the gradient. We have shown that overexpression of Dally-like protein (Dlp) causes an expansion of Gurken distribution and a loss of cell fates which are specified by high levels of epidermal growth factor receptor (Egfr) signaling. In this article, we discuss how D-Cbl mediated Egfr endocytosis and the levels of Dlp affect the shape of the Gurken gradient.     

Smiling Gurken gradient-an expansion of the Gurken gradient

Morphogen gradients provide unique positional information within a tissue. Cells that are sensitive to the concentration of the morphogen integrate this signal and develop an appropriately distinct cell fate. A morphogen gradient is usually generated by a restricted source and shaped by the speed of diffusion and stability of the signaling molecule. In addition, the availability of receptor and Heparan Sulfate Proteoglycans (HSPGs) help to shape the gradient. We have shown that over-expression of Dally-like protein (Dlp) causes an expansion of Gurken distribution and a loss of cell fates which are specified by high levels of epidermal growth factor receptor (Egfr) signaling. In this article, we discuss how D-Cbl mediated Egfr endocytosis and the levels of Dlp affect the shape of the Gurken gradient.     

The gradient of Gurken, a long-range morphogen, is directly regulated by Cbl-mediated endocytosis

The asymmetric localization of gurken mRNA and post-translational sorting mechanisms are responsible for the polar distribution of Gurken protein in Drosophila. However, endocytosis of Egfr, the receptor for Gurken in the follicle cells, also plays a role in shaping the extracellular gradient of the Gurken morphogen. Previously, we have found that mutation in the Cbl gene caused elevated Egfr signaling along the dorsoventral axis, and resulted in dorsalization phenotypes in embryos and egg shells. Here, we report that overexpression of the Cbl long isoform significantly changed Gurken distribution. Using an HRP-Gurken fusion protein, we demonstrate that internalization of the Gurken-Egfr complex depends on the activity of Cbl. Increased levels of CblL promote the internalization of this complex, leading to the reduction of free ligands. The Gurken-Egfr complex trafficks through the Rab5/Rab7 associated endocytic pathway to the lysosomal degradation compartment for signaling termination. We observe endocytic Gurken not only in the dorsal but also in the ventral follicle cells, which is, to our knowledge, the first visualization of Gurken on the ventral side of egg chambers. Our results show that Gurken travels towards the lateral/posterior of the egg chamber in the absence of Cbl, suggesting that Cbl actively regulates Gurken distribution through promoting endocytosis and subsequent degradation.     

EGFR signaling modulates synaptic connectivity via Gurken

Synaptic target selection is critical for establishing functional neuronal circuits. The mechanisms regulating target selection remain incompletely understood. We describe a role for the EGF receptor and its ligand Gurken in target selection of octopaminergic Type II neurons in the Drosophila neuromuscular system. Mutants in happyhour, a regulator of EGFR signaling, form ectopic Type II neuromuscular junctions. These ectopic innervations are due to inappropriate target selection. We demonstrate that EGFR signaling is necessary and sufficient to inhibit synaptic target selection by these octopaminergic Type II neurons, and that the EGFR ligand Gurken is the postsynaptic, muscle-derived repulsive cue. These results identify a new pathway mediating cell-type and branch-specific synaptic repulsion, a novel role for EGFR signaling in synaptic target selection, and an unexpected role for Gurken as a muscle-secreted repulsive ligand.     

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.     

Local Gurken signaling and dynamic MAPK activation during Drosophila oogenesis.

During Drosophila melanogaster oogenesis Gurken, a TGF-alpha like protein localized close to the oocyte nucleus, activates the MAPK cascade via the Drosophila EGF receptor (DER). Activation of this pathway induces different cell fates in the overlying follicular epithelium, specifying the two dorsolaterally positioned respiratory appendages and the dorsalmost cells separating them. Signal-associated internalization of Gurken protein into follicle cells demonstrates that the Gurken signal is spatially restricted and of constant intensity during mid-oogenesis. At the same time MAPK activation evolves in a spatially and temporally dynamic way and resolves into a complex pattern that presages the position of the appendages. Therefore, different dorsal follicle cell fates are not determined by a Gurken morphogen gradient. Instead they are specified by secondary signal amplification and refinement processes that integrate the Gurken signal with positive and negative feedback mechanisms generated by target genes of the DER pathway.     

Formation of a functional morphogen gradient by a passive process in tissue from the early Xenopus embryo

In early development much of the cellular diversity and pattern formation of the embryo is believed to be set up by morphogens. However, for many morphogens, including members of the TGF-beta superfamily, the mechanism(s) by which they reach distant cells is unknown. We have used immunofluorescence to detect, at single cell resolution, a morphogen gradient formed across vertebrate tissue. The TGF-beta ligand is distributed in a gradient visible up to 7 cell diameters (about 150-200 microm) from its source, and is detectable only in the extracellular space. This morphogen gradient is functional, since we demonstrate activation of a high response gene (Xeomes) and a low-response gene (Xbra) at different distances from the TGF-beta source. Expression of the high affinity type II TGF-beta receptor is necessary for detection of the gradient, but the shape of the gradient formed only depends in part on the spatial variation in the amount of receptor. Finally, we demonstrate that the molecular processes that participate in forming this functional morphogen gradient are temperature independent, since the gradient forms to a similar extent whether the cells are maintained at 4 degrees C or 23 degrees C. In contrast, TGF-beta1 internalisation by cells of the Xenopus embryo is a temperature-dependent process. Our results thus suggest that neither vesicular transcytosis nor other active processes contribute to a significant extent to the formation of the morphogen gradient we observe. We conclude that, in the model system used here, a functional morphogen gradient can be formed within a few hours by a mechanism of passive diffusion.     

The EGFR ligands Spitz and Keren act cooperatively in the Drosophila eye

The EGFR signalling cascade is responsible for coordinating a wide variety of events during Drosophila eye development. It remains something of a mystery how it is that cells are able to interpret the signal so as to choose the appropriate response from the battery of possibilities: division, differentiation, cell shape change and so on. Since the cascade is essentially linear below the receptor, different cellular responses cannot be regulated by alternative signal transduction pathways. The main diversity lies upstream, in the multiple activating ligands. Spitz, Gurken and Vein have been long studied, but little is known about the physiological functions of the fourth ligand, Keren, although various roles have been predicted based on the differences between mutants in the known ligands and those of the receptor. Here, we have isolated a mutant in the keren gene, and demonstrate that Keren does indeed participate in EGFR signalling in the eye, where it acts redundantly with Spitz to control R8 spacing, cell clustering and survival. Thus, specificity cannot be determined by ligand choice, and must instead be a consequence of cell-intrinsic factors, although we speculate that there may be some quantitative differences in signalling elicited by the two ligands.     

Formation of morphogen gradients in the Drosophila wing.

Patterning of multicellular fields requires mechanisms to coordinate developmental decisions made by populations of cells. Evidence is accumulating that the necessary information is provided by localized sources of secreted signalling proteins which act as morphogens. We review evidence that Wingless, Dpp and Hedgehog proteins act as morphogens in the developing wing of Drosophila and discuss recent work illustrating that signalling helps to shape their activity gradients by regulating ligand distribution and by modulating the responsiveness of target cells. These studies suggest that there is more to being a morphogen than formation of a ligand gradient by passive diffusion.     

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.     

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.     

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.     

Gurken, a TGF-alpha-like protein involved in axis determination in Drosophila, directly binds to the EGF-receptor homolog Egfr

The establishment of axial polarity in the Drosophila egg and embryo depends on intercellular communication between two cell types in the ovary, the germline, and the soma. The genes gurken and egfr encode two essential players of this communication pathway. Gurken protein, a TGF-alpha-like molecule, is expressed in the germline, while the EGF-receptor homolog, Egfr, is expressed in the somatic cells of the ovary. Using the yeast two-hybrid system we show here, for the first time, that Gurken protein directly binds to the extracellular domain of Egfr. This direct physical association requires the presence of an intact EGF motif within Gurken protein. Furthermore, we provide evidence that this characteristic motif may be sufficient for interaction with the receptor, at list in vitro. Our results firmly establish Gurken as the germline ligand of Drosophila Egfr.     

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.     

Misexpression of argos, an inhibitor of EGFR signaling in oogenesis, leads to the production of bicephalic, ventralized, and lateralized Drosophila melanogaster eggs

Epidermal growth factor receptor (EGFR) signaling pathways are frequently involved in generating cell fate diversity in a number of organisms. During anterior-posterior and dorso-ventral polarity in the Drosophila egg chamber and eggshell, EGFR signaling leads to a number of determinative events in the follicle cell layer. A high level of Gurken signal leads to the expression of argos in dorsal midline cells. Lateral follicle cells, receiving a lower level of Gurken signal, can continue to express the Broad-Complex (BR-C) and differentiate into cells which produce chorionic appendages. Misexpression of argos in mid-oogenesis causes the midline cells to retain expression of BR-C, resulting in a single fused large appendage. Evidence that argos can directly repress Gurken-induced EGFR signaling is seen when premature expression of argos is induced earlier in oogenesis. It represses the Gurken signal at stage 5-6 of oogenesis which determines posterior follicle cells and occasionally leads to eggs with anteriors at both ends. We propose that the Gurken signal at stage 9 of oogenesis induces follicle cells to take on two fates, dorsal midline and lateral, each producing different parts of the eggshell and that argos is one of the key downstream genes required to select between these two fates.     

Multiple EGFR ligands participate in guiding migrating border cells

Cell migration is an important feature of embryonic development as well as tumor metastasis. Border cells in the Drosophila ovary have emerged as a useful in vivo model for uncovering the molecular mechanisms that control many aspects of cell migration including guidance. It was previously shown that two receptor tyrosine kinases, epidermal growth factor receptor (EGFR) and PDGF- and VEGF-related receptor (PVR), together contribute to border cell migration. Whereas the ligand for PVR, PVF1, is known to guide border cells, it is unclear which of the four activating EGFR ligands function in this process. We developed an assay to detect the ability of secreted factors to reroute migrating border cells in vivo and tested the activity of EGFR ligands compared to PVF1. Two ligands, Keren and Spitz, guided border cells whereas the other ligands, Gurken and Vein, did not. In addition, only Keren and Spitz were expressed at the appropriate stage in the oocyte, the target of border cell migration. Therefore, a complex combination of EGFR and PVR ligands together guide border cells to the oocyte.     

Dpp gradient formation by dynamin-dependent endocytosis: receptor trafficking and the diffusion model

Developing cells acquire positional information by reading the graded distribution of morphogens. In Drosophila, the Dpp morphogen forms a long-range concentration gradient by spreading from a restricted source in the developing wing. It has been assumed that Dpp spreads by extracellular diffusion. Under this assumption, the main role of endocytosis in gradient formation is to downregulate receptors at the cell surface. These surface receptors bind to the ligand and thereby interfere with its long-range movement. Recent experiments indicate that Dpp spreading is mediated by Dynamin-dependent endocytosis in the target tissue, suggesting that extracellular diffusion alone cannot account for Dpp dispersal. Here, we perform a theoretical study of a model for morphogen spreading based on extracellular diffusion, which takes into account receptor binding and trafficking. We compare profiles of ligand and surface receptors obtained in this model with experimental data. To this end, we monitored directly the pool of surface receptors and extracellular Dpp with specific antibodies. We conclude that current models considering pure extracellular diffusion cannot explain the observed role of endocytosis during Dpp long-range movement.     

The Wingless morphogen gradient is established by the cooperative action of Frizzled and Heparan Sulfate Proteoglycan receptors

We have examined the respective contribution of Heparan Sulfate Proteoglycans (HSPGs) and Frizzled (Fz) proteins in the establishment of the Wingless (Wg) morphogen gradient. From the analysis of mutant clones of sulfateless/N-deacetylase-sulphotransferase in the wing imaginal disc, we find that lack of Heparan Sulfate (HS) causes a dramatic reduction of both extracellular and intracellular Wg in receiving cells. Our studies, together with others [Kirkpatrick, C.A., Dimitroff, B.D., Rawson, J.M., Selleck, S.B., 2004. Spatial regulation of Wingless morphogen distribution and signalling by Dally-like protein. Dev. Cell (in press)], reveals that the Glypican molecule Dally-like Protein (Dlp) is associated with both negative and positive roles in Wg short- and long-range signaling, respectively. In addition, analyses of the two Fz proteins indicate that the Fz and DFz2 receptors, in addition to transducing the signal, modulate the slope of the Wg gradient by regulating the amount of extracellular Wg. Taken together, our analysis illustrates how the coordinated activities of HSPGs and Fz/DFz2 shape the Wg morphogen gradient.     

CSN5/Jab1 mutations affect axis formation in the Drosophila oocyte by activating a meiotic checkpoint

The COP9 signalosome (CSN) is linked to signaling pathways and ubiquitin-dependent protein degradation in yeast, plant and mammalian cells, but its roles in Drosophila development are just beginning to be understood. We show that during oogenesis CSN5/JAB1, one subunit of the CSN, is required for meiotic progression and for establishment of both the AP and DV axes of the Drosophila oocyte. The EGFR ligand Gurken is essential for both axes, and our results show that CSN5 mutations block the accumulation of Gurken protein in the oocyte. CSN5 mutations also cause the modification of Vasa, which is known to be required for Gurken translation. This CSN5 phenotype - defective axis formation, reduced Gurken accumulation and modification of Vasa - is very similar to the phenotype of the spindle-class genes that are required for the repair of meiotic recombination-induced, DNA double-strand breaks. When these breaks are not repaired, a DNA damage checkpoint mediated by mei-41 is activated. Accordingly, the CSN5 phenotype is suppressed by mutations in mei-41 or by mutations in mei-W68, which is required for double strand break formation. These results suggest that, like the spindle-class genes, CSN5 regulates axis formation by checkpoint-dependent, translational control of Gurken. They also reveal a link between DNA repair, axis formation and the COP9 signalosome, a protein complex that acts in multiple signaling pathways by regulating protein stability.