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.     

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.     

The interpretation of morphogen gradients

Morphogens act as graded positional cues that control cell fate specification in many developing tissues. This concept, in which a signalling gradient regulates differential gene expression in a concentration-dependent manner, provides a basis for understanding many patterning processes. It also raises several mechanistic issues, such as how responding cells perceive and interpret the concentration-dependent information provided by a morphogen to generate precise patterns of gene expression and cell differentiation in developing tissues. Here, we review recent work on the molecular features of morphogen signalling that facilitate the interpretation of graded signals and attempt to identify some emerging common principles.     

Models for cell differentiation and generation of polarity in diffusion-governed morphogenetic fields

Models based on molecular mechanisms are presented for pattern formation in developing organisms. It is assumed that there exists a diffusion governed gradient in the morphogenetic field. It is shown that cellular differentiation and the subsequent pattern formation result from the interaction of the diffusing morphogen with the genetic regulatory mechanism of cells. In a second stage it is shown that starting from a homogeneous distribution of morphogen, polarity can be generated spontaneously in the morphogenetic field giving rise to the establishment of a gradient. The stability of these gradients is demonstrated. The onset of a morphogenetic gradient and pattern formation are combined in a single coherent model. Size invariance and its biological implications are discussed.     

Pattern formation: fruiting body morphogenesis in Myxococcus xanthus

When Myxococcus xanthus cells are exposed to starvation, they respond with dramatic behavioral changes. The expansive swarming behavior stops and the cells begin to aggregate into multicellular fruiting bodies. The cell-surface-associated C-signal has been identified as the signal that induces aggregation. Recently, several of the components in the C-signal transduction pathway have been identified and behavioral analyses are beginning to reveal how the C-signal modulates cell behavior. Together, these findings provide a framework for understanding how a cell-surface-associated morphogen induces pattern formation.     

Drosophila embryo syncytial blastoderm cellular architecture and morphogen gradient dynamics: Is there a correlation?

During embryo development in many metazoan animals, the first differentiated cell type to form is an epithelial cell. This epithelial layer is modified by developmental cues of body axes formation to give rise to various tissues. The cells that arise are mesenchymal in nature and are a source of other tissue types. This epithelial to mesenchymal transition is used for tissue type formation and also seen in diseases such as cancer. Here we discuss recent findings on the cellular architecture formation in the Drosophila embryo and how it affects the developmental program of body axes formation. In particular these studies suggest the presence of compartments around each nucleus in a common syncytium. Despite the absence of plasma membrane boundaries, each nucleus not only has its own endoplasmic reticulum and Golgi complex but also its own compartmentalized plasma membrane domain above it. This architecture is potentially essential for morphogen gradient restriction in the syncytial Drosophila embryo. We discuss various properties of the dorso-ventral and the antero-posterior morphogen gradients in the Drosophila syncytium, which are likely to depend on the syncytial architecture of the embryo.     

When are cellular oscillators sufficient for sequential segmentation?

Sequential segmentation during embryogenesis involves the generation of a repeated pattern along the embryo, which is concurrently undergoing axial elongation by cell division. Most mathematical models of sequential segmentation involve inherent cellular oscillators, acting as a segmentation clock. The cellular oscillation is assumed to be governed by the cell's physiological age or by its interaction with an external morphogen gradient. Here, we address the issue of when cellular oscillators alone are sufficient for predicting segmentation, and when a morphogen gradient is required. The key to resolving this issue lies in how cells determine positional information in the model - this is directly related to the distribution of cell divisions responsible for axial elongation. Mathematical models demonstrate that if axial elongation occurs through cell divisions restricted to the posterior end of the unsegmented region, a cell can obtain its positional information from its physiological age, and therefore cellular oscillators will suffice. Alternatively, if axial elongation occurs through cell divisions distributed throughout the unsegmented region, then positional information can be obtained through another mechanism, such as a morphogen gradient. Two alternative ways to establish a morphogen gradient in tissue with distributed cell divisions are presented - one with diffusion and the other without diffusion. Our model produces segment polarity and a distribution of segment size from the anterior-to-posterior ends, as observed in some systems. Furthermore, the model predicts segment deletions when there is an interruption in cell division, just as seen in heat shock experiments, as well as the growth and final shrinkage of the presomitic mesoderm during somitogenesis.     

Pattern formation: fruiting body morphogenesis in Myxococcus xanthus

When Myxococcus xanthus cells are exposed to starvation, they respond with dramatic behavioral changes. The expansive swarming behavior stops and the cells begin to aggregate into multicellular fruiting bodies. The cell-surface-associated C-signal has been identified as the signal that induces aggregation. Recently, several of the components in the C-signal transduction pathway have been identified and behavioral analyses are beginning to reveal how the C-signal modulates cell behavior. Together, these findings provide a framework for understanding how a cell-surface-associated morphogen induces pattern formation.     

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.     

Establishment of global patterns of planar polarity during growth of the Drosophila wing epithelium

Epithelial tissues develop planar polarity that is reflected in the global alignment of hairs and cilia with respect to the tissue axes. The planar cell polarity (PCP) proteins form asymmetric and polarized domains across epithelial junctions that are aligned locally between cells and orient these external structures. Although feedback mechanisms can polarize PCP proteins intracellularly and locally align polarity between cells, how global PCP patterns are specified is not understood. It has been proposed that the graded distribution of a biasing factor could guide long-range PCP. However, we recently identified epithelial morphogenesis as a mechanism that can reorganize global PCP patterns; in the Drosophila pupal wing, oriented cell divisions and rearrangements reorient PCP from a margin-oriented pattern to one that points distally. Here, we use quantitative image analysis to study how PCP patterns first emerge in the wing. PCP appears during larval growth and is spatially oriented through the activities of three organizer regions that control disc growth and patterning. Flattening morphogen gradients emanating from these regions does not reduce intracellular polarity but distorts growth and alters specific features of the PCP pattern. Thus, PCP may be guided by morphogenesis rather than morphogen gradients.     

Forming and Interpreting Gradients in the Early Xenopus Embryo

The amphibian embryo provides a powerful model system to study morphogen gradients because of the ease with which it is possible to manipulate the early embryo. In particular, it is possible to introduce exogenous sources of morphogen, to follow the progression of the signal, to monitor the cellular response to induction, and to up- or down-regulate molecules that are involved in all aspects of long-range signaling. In this article, I discuss the evidence that gradients exist in the early amphibian embryo, the way in which morphogens might traverse a field of cells, and the way in which different concentrations of morphogens might be interpreted to activate the expression of different genes.The idea that a morphogen gradient activates the expression of different genes at different concentrations was perhaps stated most clearly by Wolpert''s French flag model, in which a graded signal activates the expression of “blue,” “white,” and “red” genes at high, intermediate, and low concentrations (Wolpert 1969). Since that original work, great progress has been made in identifying morphogens and their target genes and it is now clear that the spatial pattern of gene expression in the developing embryo is frequently established by graded signals of this sort. But many questions remain, and in particular little is known about how gradients are established in the embryo with the necessary precision and how cells interpret different concentrations of morphogen to activate different genes. I discuss these issues with respect to mesoderm induction in the developing amphibian embryo.     

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.     

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.     

Space-dependent cell determination under the control of a morphogen gradient

A model is proposed for space-dependent cell determination under the influence of a morphogen gradient. It provides an explanation of how groups of cells can be programmed in a particular direction and how a jump from one determination stage to the next can occur between them even though the controlling signal is of a smoothly graded morphogen concentration. Together with an earlier proposed mechanism for pattern formation, these models offer a complete system for the generation and interpretation of positional information. Each member of a set of structure-controlling genes is assumed to feed back onto its own activation such that a gene, once activated, remains in the activated state. A repressor, however, is produced by any activated gene of this set. This assures that only one gene of this set is active in one cell at any one time. A selective activation of a particular gene is possible if (i) the morphogen competes with the gene-produced, non-diffusible repressor, (ii) the feedback loops have some overlap and (iii) a hierarchy exists among the structure-controlling genes. The kinetics of this determination have all the properties demanded earlier from a study of the early insect development: It proceeds stepwise from determination for more anterior to more posterior structures until the gene that is activated corresponds to the local gradient level. A more anterior structure will be formed if the gradient is destroyed before the final determination level is reached. A more posterior structure will be formed after an additional increase of the morphogen concentration. After completion of the determination, the repressor concentration in each cell depends on which gene has become activated and it can be made roughly proportional to the morphogen concentration which the cell has seen. Therefore, a stable parameter (positional value) becomes available which can be used for further developmental decisions.     

A Dictyostelium morphogen that is essential for stalk cell formation is generated by a subpopulation of prestalk cells

The stalk cell differentiation inducing factor (DIF) has the properties required of a morphogen responsible for pattern regulation during the pseudoplasmodial stage of Dictyostelium development. It induces prestalk cell formation and inhibits prespore cell formation, but there is as yet no strong evidence for a morphogenetic gradient of DIF. We have measured DIF accumulation by monolayers of isolated prestalk and prespore cells in an attempt to provide evidence for such a gradient. DIF is accumulated in the largest quantities by a subpopulation of prestalk cells that specifically express the DIF-inducible genes pDd56 and pDd26. Since it has been shown recently that cells that express pDd56 are localized in the central core of the prestalk cell region of the pseudoplasmodia, our current results suggest a morphogenetic gradient generated by this region.     

Morphogens in chick limb development

Retinoic acid is a good candidate for a morphogen in chick limb bud development. The challenge now is to determine how retinoic acid interacts with limb bud cells and how the retinoic acid signal is integrated with other signals to mould and pattern the developing limb.     

Patterned cell determination in a plant tissue: the secondary phloem of trees

The secondary vascular tissues (xylem and phloem) of woody plants originate from a vascular cambium and develop as radially oriented files of cells. The secondary phloem is composed of three or four cell types, which are organised into characteristic recurrent cellular sequences within the radial cell files of this tissue. There is a gradient of auxin (indole acetic acid) across both the cambium and the immediately postmitotic cells within the xylem and phloem domains, and it is believed that this morphogen, probably in concert with other morphogenic factors, is closely associated with the determination and differentiation of the different cells types in each tissue. A hypothesis is developed that, in conjunction with the positional values conferred by the graded radial distribution of morphogen, cell divisions at particular positions within the cambium are sufficient to determine not only each of the phloem cell types but also their recurrent pattern of differentiation within each radial cell file.