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.     

Membrane-Associated Non-Receptors and Morphogen Gradients

A previously investigated basic model (System B) for the study of signaling morphogen gradient formation that allows for reversible binding of morphogens (aka ligands) with signaling receptors, degradation of bound morphogens and diffusion of unbound morphogens is extended to include the effects of membrane-bound non-signaling molecules (or non-receptors for short) such as proteoglycans that bind reversibly with the same morphogens and degrade them. Our main goal is to delineate the effects of the presence of non-receptors on the existence and properties of the steady-state concentration gradient of signaling ligand–receptor complexes. Stability of the steady-state morphogen gradients is established and the time to reach steady-state behavior after the onset of morphogen production will be analyzed. The theoretical findings offer explanations for observations reported in several previous experiments on Drosophila wing imaginal discs.     

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.     

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.     

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 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.     

A model of morphogenetic pattern formation

A model for the morphogenetic movement of surfaces composed of cellular monolayers is proposed. The cells are presumed joined at their lateral surfaces. An otherwise unspecified substance called a "morphogen" is introduced which is the agent of change in the individual cell (or cell-like region). The distribution of these cellular deformations define a surface (the middle surface, through the middle of the cell heights) via equations given for the Gauss and Mean curvatures of the surface defined at each point. The Gauss curvature as a function of the morphogen level determines the metric of the surface "g(u, v)" in conformal co-ordinates u, v. A unique equation for the morphogen distribution over the survace is presented which has the property of size invariance, that is, the model "regulates" without need of further arguments. The two resulting coupled equations for the metric and the morphogen, eqns (4) and (2), both non-linear equations, are to be solved self-consistently, once the individual cell deformation as a function of morphogen is given. The surface geometry determines the morphogen distribution, and the morphogen distribution in turn affects the surface geometry. Extension of the model to two or more morphogens is straightforward, and the key property of "regulation" or size invariance of the model is retained. Numerical integration of the two coupled equations is carried out in the case of axial symmetry, and the results presented by the case that individual cells deform by changing the ratio of their apical to basal areas, as well as their heights. Gastrulation in small regulating holoblastic eggs (e.g. starfish, sea urchin and amphioxus) is discussed in light of the present model.     

Three Drosophila EXT genes shape morphogen gradients through synthesis of heparan sulfate proteoglycans

The signaling molecules Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg) function as morphogens and organize wing patterning in Drosophila. In the screen for mutations that alter the morphogen activity, we identified novel mutants of two Drosophila genes, sister of tout-velu (sotv) and brother of tout-velu (botv), and new alleles of tout-velu (ttv). The encoded proteins of these genes belong to an EXT family of proteins that have or are closely related to glycosyltransferase activities required for biosynthesis of heparan sulfate proteoglycans (HSPGs). Mutation in any of these genes impaired biosynthesis of HSPGs in vivo, indicating that, despite their structural similarity, they are not redundant in the HSPG biosynthesis. Protein levels and signaling activities of Hh, Dpp and Wg were reduced in the cells mutant for any of these EXT genes to a various degree, Wg signaling being the least sensitive. Moreover, all three morphogens were accumulated in the front of EXT mutant cells, suggesting that these morphogens require HSPGs to move efficiently. In contrast to previous reports that ttv is involved exclusively in Hh signaling, we found that ttv mutations also affected Dpp and Wg. These data led us to conclude that each of three EXT genes studied contribute to Hh, Dpp and Wg morphogen signaling. We propose that HSPGs facilitate the spreading of morphogens and therefore, function to generate morphogen concentration gradients.     

Genetics of morphogen gradients

The organization of cells and tissues is controlled by the action of 'form-giving' signalling molecules, or morphogens, which pattern a developmental field in a concentration-dependent manner. As the fate of each cell in the field depends on the level of the morphogen signal, the concentration gradient of the morphogen prefigures the pattern of development. In recent years, molecular genetic studies in Drosophila melanogaster have allowed tremendous progress in understanding how morphogen gradients are formed and maintained, and the mechanism by which receiving cells respond to the gradient.     

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.     

Mathematical Model of the Formation of Morphogen Gradients Through Membrane-Associated Non-receptors

The importance of morphogens is a central concept in developmental biology. Multiple-fate patterning and the robustness of the morphogen gradient are essential for embryo development. The ways by which morphogens diffuse from a local source to form long distance gradients can differ from one morphogen to the other, and for the same morphogen in different organs. This paper will study the mechanism by which morphogens diffuse through the aid of membrane-associated non-receptors and will investigate how the membrane-associated non-receptors help the morphogen to form long distance gradients and to achieve good robustness. Such a mechanism has been reported for some morphogens that are rapidly turned over. We will establish a set of reaction-diffusion equations to model the dynamical process of morphogen gradient formation. Under the assumption of rapid morphogen degradation, we discuss the existence, uniqueness, local stability, approximation solution, and the robustness of the steady-state gradient. The results in this paper show that when the morphogen is rapidly turned over, diffusion of the morphogen through membrane-associated non-receptors is a possible strategy to form a long distance multiple-fate gradient that is locally stable and is robust against the changes in morphogen synthesis rate.     

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.     

A conserved mechanism of Hedgehog gradient formation by lipid modifications

Members of the Hedgehog (Hh) family of proteins are conserved morphogens that modulate cell fates in target tissues in different developmental systems. Dysregulation of Hh signaling results in a wide range of human diseases. The mature Hh is modified by lipids in two places, with palmitate at the N-terminus and cholesterol at the C-terminus. The lipid modifications are essential to the proper secretion and spreading of the morphogen throughout the extracellular matrix, interacting with heparan sulfate proteoglycans. However, the role of lipid modifications in regulating the range and activity of Hh proteins remains controversial. Here, we aim to resolve this issue by providing a model that is consistent with current and past reports. We propose that the cholesterol moiety functions to restrict the dilution and deregulated spread of the morphogen in the extracellular space.     

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.     

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.     

Modulation of developmental signals by endocytosis: different means and many ends

Recent advances have highlighted the importance of endocytic processes in regulating the activity and distribution of developmental signals. Classically, signalling is downregulated by endocytosis and subsequent trafficking to lysosomes (e.g. Notch, Hedgehog, Roundabout). However, endocytosis can also have a positive role in signalling. For example, endocytosis of Delta, the ligand of Notch, is needed for activation of the signal. In the case of signalling by Hedgehog, endocytic trafficking segregates an inhibitory receptor (Patched) from the positive effector (Smoothened). Endosomes could also be the site where signalling is activated (e.g. transforming growth factor beta). Finally, endocytosis could power the transport of morphogens along epithelia.     

Morphogens and cell survival during development

The notion of "morphogens" is an important one in developmental biology. By definition, a morphogen is a molecule that emanates from a specific set of cells that is present in a concentration gradient and that specifies the fate of each cell along this gradient. The strongest candidate morphogens are members of the transforming growth factor-beta (TGF-beta), Hedgehog (Hh), and Wnt families. While these morphogens have been extensively described as differentiation inducers, some reports also suggest their possible involvement in cell death and cell survival. It is frequently speculated that the cell death induction that is found associated with experimental removal of morphogens is the manifestation of abnormal differentiation signals. However, several recent reports have raised controversy about this death by default, suggesting that cell death regulation is an active process for shaping tissues and organs. In this review, we will present morphogens, with a specific emphasis on Sonic Hedgehog, a mammalian member of the Hh family, not as a positive regulators of cell differentiation but as key regulators of cell survival.     

Problems and paradigms: Morphogens and pattern formation

Morphogen gradient theories have enjoyed considerable popularity since the beginning of this century, but conclusive evidence for a role of morphogens in controlling multicellular development has been elusive. Recently, work on three secreted signalling proteins, Activin in Xenopus, and Wingless and Dpp in Drosophila, has stongly suggested that these proteins function as morphogens. In order to define a factor as a morphogen, it is necessary to show firstly, that it has a direct effect on target cells and secondly, that it affects the development of target cells in a concentration-dependent manner. With these criteria in mind, the evidence available for a variety of proposed morphogens is discussed. While the evidence is not conclusive in most of the cases considered, there is a strong case in favour of the three proteins mentioned above, which suggests that morphogens are potentially of general importance in controlling the development of multicellular organisms.