Morphogens, their identification and regulation

During the course of development, cells of many tissues differentiate according to the positional information that is set by the concentration gradients of morphogens. Morphogens are signaling molecules that emanate from a restricted region of a tissue and spread away from their source to form a concentration gradient. As the fate of each cell in the field depends on the concentration of the morphogen signal, the gradient prefigures the pattern of development. In this article, we describe how morphogens and their functions have been identified and analyzed, focusing on model systems that have been extensively studied.     

Morphing into cancer: the role of developmental signaling pathways in brain tumor formation

Morphogens play a critical role in most aspects of development, including expansion and patterning of the central nervous system. Activating germline mutations in components of the Hedgehog and Wnt pathways have provided evidence for the important roles morphogens play in the genesis of brain tumors such as cerebellar medulloblastoma. In addition, aberrant expression of transforming growth factor-beta (TGF-beta) superfamily members has been demonstrated to contribute to progression of malignant gliomas. This review summarizes our current knowledge about the roles of morphogens in central nervous system tumorigenesis.     

Longitudinal axon guidance

Our knowledge about molecular mechanisms underlying axon guidance along the antero-posterior axis in contrast to the dorso-ventral axis of the developing nervous system is very limited. During the past two years in vitro and in vivo studies have indicated that morphogens have a role in longitudinal axon guidance. Morphogens are secreted proteins that act in a concentration-dependent manner on susceptible groups of precursor cells and induce their differentiation to a specific cell fate. Thus, gradients of morphogens are responsible for the appropriate patterning of the nervous system during early phases of neural development. Therefore, it was surprising to find that gradients of two of these morphogens, Wnt4 and Shh, can be re-used for longitudinal axon guidance during later stages of nervous system development.     

Morphogen gradients. A question of time or concentration?

Morphogens are secreted proteins that organize surrounding tissues into distinct territories and are thought to act as a function of a threshold of their concentration. Although it has been demonstrated that morphogens act directly on the cells and do not rely on secondary signalling relays, intracellular sequential induction mechanisms, which are dependent on a simple signalling instruction, have not been excluded. Here, we present an alternative model to account for the organizing properties of morphogens, and propose that initial exposure to signalling changes cell context, which in combination with continuing morphogen activity, results in the expression of novel targets.     

Carrier of Wingless (Cow), a Secreted Heparan Sulfate Proteoglycan,Promotes Extracellular Transport of Wingless

Morphogens are signaling molecules that regulate growth and patterning during development by forming a gradient and activating different target genes at different concentrations. The extracellular distribution of morphogens is tightly regulated, with the Drosophila morphogen Wingless (Wg) relying on Dally-like (Dlp) and transcytosis for its distribution. However, in the absence of Dlp or endocytic activity, Wg can still move across cells along the apical (Ap) surface. We identified a novel secreted heparan sulfate proteoglycan (HSPG) that binds to Wg and promotes its extracellular distribution by increasing Wg mobility, which was thus named Carrier of Wg (Cow). Cow promotes the Ap transport of Wg, independent of Dlp and endocytosis, and this function addresses a previous gap in the understanding of Wg movement. This is the first example of a diffusible HSPG acting as a carrier to promote the extracellular movement of a morphogen.     

Understanding morphogenetic growth control -- lessons from flies

Morphogens are secreted signalling molecules that control the patterning and growth of developing organs. How morphogens regulate patterning is fairly well understood; however, how they control growth is less clear. Four principal models have been proposed to explain how the morphogenetic protein Decapentaplegic (DPP) controls the growth of the wing imaginal disc in the fly. Recent studies in this model system have provided a wealth of experimental data on growth and DPP gradient properties, as well as on the interactions of DPP with other signalling pathways. These findings have allowed a more precise formulation and evaluation of morphogenetic growth models. The insights into growth control by the DPP gradient will also be useful for understanding other morphogenetic growth systems.     

Mechanisms of Hedgehog gradient formation and interpretation

Morphogens are molecules that spread from localized sites of production, specifying distinct cell outcomes at different concentrations. Members of the Hedgehog (Hh) family of signaling molecules act as morphogens in different developmental systems. If we are to understand how Hh elicits multiple responses in a temporally and spatially specific manner, the molecular mechanism of Hh gradient formation needs to be established. Moreover, understanding the mechanisms of Hh signaling is a central issue in biology, not only because of the role of Hh in morphogenesis, but also because of its involvement in a wide range of human diseases. Here, we review the mechanisms affecting the dynamics of Hh gradient formation, mostly in the context of Drosophila wing development, although parallel findings in vertebrate systems are also discussed.     

Morphogen gradient formation and vesicular trafficking

Morphogens are secreted signaling molecules which form spatial concentration gradients while moving away from a restricted source of production. A simple model of gradient formation postulates that the morphogens dilute as they diffuse between cells. In this review we discuss recent data supporting the idea that movement of the morphogen could also occur via vesicular trafficking through the cells. We explore the implications of these results for the control of gradient formation and the determination of the gradient slope which ultimately encodes the coordinates of positional information.     

The interplay between morphogens and tissue growth

Morphogens are conserved, secreted signalling molecules that regulate the size, shape and patterning of animal tissues and organs. Recent experimental evidence has emphasized the fundamental role of tissue growth in expanding the expression domains of morphogens and their target genes, in generating morphogen gradients and in modulating the response of cells to morphogens. Moreover, the classic view of how morphogens, particularly through their concentration gradient, regulate tissue size during development has been revisited recently. In this review, we discuss how morphogens and tissue growth affect each other, and we attempt to integrate genetic and molecular evidence from vertebrate and invertebrate model systems to put forward the idea that the interaction between growth and morphogens is a general feature of highly proliferative tissues.     

Visualizing long-range movement of the morphogen Xnr2 in the Xenopus embryo

One way in which cells acquire positional information during embryonic development is by measuring the local concentration of a signaling factor, or morphogen, that is secreted by an organizing center . The ways in which morphogen gradients are established, particularly in vertebrates, remain obscure, although various suggestions have been made for the mechanisms by which signaling molecules traverse fields of cells. These include simple diffusion, "cytonemes", filopodia, "argosomes", and "transcytosis". In this study, we use a functional EGFP-tagged ligand to visualize long-range signaling in the Xenopus embryo in real time. Our results show that the TGF-beta family member Xnr2 is secreted efficiently from embryonic cells, and a new method of tissue recombination allows us to investigate the way in which the morphogen traverses multiple cell diameters. This reveals that Xnr2 exerts long-range effects by diffusing rapidly through the extracellular milieu of nonexpressing cells. No evidence has been obtained for long-range signaling through cytonemes, filopodia, argosomes, or transcytosis. In demonstrating that long-range signaling in the early Xenopus embryo occurs by diffusion rather than by these alternative routes, our results suggest that different morphogens in different developmental contexts use different means of transport.     

APC dosage effects in tumorigenesis and stem cell differentiation

It is well established that concentration gradients of signaling molecules (the so-called "morphogens") organize and pattern tissues in developing animals. In particular, studies in Drosophila and different vertebrates have shown that gradients of the Wnt, Hedgehog (Hh) and transforming growth factor-beta (TGF-beta) families of morphogens play critical roles in limb patterning. Morphogens are often expressed in organizing centres and can act over a long range to coordinate the patterning of an entire field of cells. These observations imply that exposure to different concentrations of these diffusible factors may trigger differential cellular responses. In order to study these dosage-dependent Wnt/beta-catenin signaling effects, we have generated several hypomorphic mutant alleles at the mouse Apc locus and studied their cellular and phenotypic outcomes in stem cell renewal and differentiation, and in tumorigenesis. The results clearly show that Apc mutations differentially affect the capacity of stem cells to differentiate in a dosage-dependent fashion. Likewise, different Apc mutations (and the corresponding Wnt signaling dosages) confer different degrees of susceptibility to tumorigenesis in the corresponding mouse models. These results have implications for the understanding of the molecular and cellular basis of tumor initiation by defects in the Wnt pathway. We propose a model in which adult somatic stem cell compartments are characterized by tissue-specific beta-catenin threshold levels for cell proliferation, differentiation and apoptosis. Different APC mutations will result in different levels of beta-catenin signaling, thus conferring different degrees of tumor susceptibility in different tissues. Hence, beta-catenin dosage-dependent effects may not only explain how a single pathway is involved in the development and homeostasis of different tissues, but also its pleiotrophic role in tumorigenesis.     

Wnts as morphogens? The view from the wing of Drosophila

Morphogens are diffusible signalling molecules that pattern cellular fields by setting up differential gene expression in a concentration-dependent manner. Members of the Wnt family of signalling molecules are generally considered to be classical morphogens. However, a close analysis of their activity indicates that they do not fulfil all of the critera that are associated with the classical definition.     

A microfluidic system for investigation of extravascular transport and cellular uptake of drugs in tumors

Three-dimensional (3D) tumor models have been established in various microfluidic systems for drug delivery and resistance studies in vitro. However, one of the main drawbacks of these models is non-uniform distribution of cells, leaving regions with very low cell density within the 3D structures. As a result, molecular diffusion in the cell compartments is faster than that observed in solid tumors. To solve this problem, we developed a new technique for preparation of 3D tumor models in vitro. It was based on a microfluidic device containing three parallel channels separated by narrowly spaced posts. Tumor cells were loaded into the central channel at high density. To test the system, B16.F10 melanoma cells were perfusion-cultured overnight and the resulting 3D structure was characterized in terms of viability, density, and morphology of cells as well as transport properties of small fluorescent molecules. Immediately upon loading of tumor cells, the cell density was comparable to those observed in B16.F10 tumor tissues in vivo; and the viability of tumor cells was maintained through the overnight culture. The tumor model displayed low extracellular space and high resistance to diffusion of small molecules. For membrane-permeant molecules (e.g., Hoechst 33342), the rate of interstitial penetration was extremely slow, compared to membrane-impermeant molecules (e.g., sodium fluorescein). This versatile tumor model could be applied to in vitro studies of transport and cellular uptake of drugs and genes.     

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.     

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.     

Bicoid gradient formation mechanism and dynamics revealed by protein lifetime analysis

Embryogenesis relies on instructions provided by spatially organized signaling molecules known as morphogens. Understanding the principles behind morphogen distribution and how cells interpret locally this information remains a major challenge in developmental biology. Here, we introduce morphogen‐age measurements as a novel approach to test models of morphogen gradient formation. Using a tandem fluorescent timer as a protein age sensor, we find a gradient of increasing age of Bicoid along the anterior–posterior axis in the early Drosophila embryo. Quantitative analysis of the protein age distribution across the embryo reveals that the synthesis–diffusion–degradation model is the most likely model underlying Bicoid gradient formation, and rules out other hypotheses for gradient formation. Moreover, we show that the timer can detect transitions in the dynamics associated with syncytial cellularization. Our results provide new insight into Bicoid gradient formation and demonstrate how morphogen‐age information can complement knowledge about movement, abundance, and distribution, which should be widely applicable to other systems.     

Wingless gradient formation in the Drosophila wing

BACKGROUND: Secreted signaling proteins of the Wingless (Wg)/Wnt, Hedgehog and bone morphogenetic protein (BMP)/Decapentaplegic (Dpp) families function as morphogens to control growth and pattern formation during development. Although these proteins have been shown to act directly on distant cells in the developing limbs of the fruit fly Drosophila, little is known about how ligand gradients form in vivo. Wg protein is found in vesicles in Wg-responsive cells in the embryo and imaginal discs. It has been proposed that Wg may be transported by a vesicle-mediated mechanism. RESULTS: A novel method to visualize extracellular Wg protein was used to show that Wg forms an unstable gradient on the basolateral surface of the wing imaginal disc epithelium. Wg movement did not require internalization by dynamin-mediated endocytosis. Dynamin activity was, however, required for Wg secretion. By reversibly blocking Wg secretion, we found that Wg moves rapidly to form a long-range extracellular gradient. CONCLUSIONS: The Wg morphogen gradient forms by rapid movement of ligand through the extracellular space, and depends on continuous secretion and rapid turnover. Endocytosis is not required for Wg movement, but contributes to shaping the gradient by removing extracellular Wg. We propose that the extracellular Wg gradient forms by diffusion.