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.     

Self-organized shuttling: generating sharp dorsoventral polarity in the early Drosophila embryo

Morphogen gradients pattern tissues and organs during development. When morphogen production is spatially restricted, diffusion and degradation are sufficient to generate sharp concentration gradients. It is less clear how sharp gradients can arise within the source of a broadly expressed morphogen. A recent solution relies on localized production of an inhibitor outside the domain of morphogen production, which effectively redistributes (shuttles) and concentrates the morphogen within its expression domain. Here, we study how a sharp gradient is established without a localized inhibitor, focusing on early dorsoventral patterning of the Drosophila embryo, where an active ligand and its inhibitor are concomitantly generated in a broad ventral domain. Using theory and experiments, we show that?a sharp?Toll activation gradient is produced through "self-organized shuttling," which dynamically relocalizes inhibitor production to lateral regions, followed by inhibitor-dependent ventral shuttling of the activating ligand Sp?tzle. Shuttling may represent?a general paradigm for patterning early embryos. PAPERFLICK:     

Notochord-derived Shh concentrates in close association with the apically positioned basal body in neural target cells and forms a dynamic gradient during neural patterning

Sonic hedgehog (Shh) ligand secreted by the notochord induces distinct ventral cell identities in the adjacent neural tube by a concentration-dependent mechanism. To study this process, we genetically engineered mice that produce bioactive, fluorescently labeled Shh from the endogenous locus. We show that Shh ligand concentrates in close association with the apically positioned basal body of neural target cells, forming a dynamic, punctate gradient in the ventral neural tube. Both ligand lipidation and target field response influence the gradient profile, but not the ability of Shh to concentrate around the basal body. Further, subcellular analysis suggests that Shh from the notochord might traffic into the neural target field by means of an apical-to-basal-oriented microtubule scaffold. This study, in which we directly observe, measure, localize and modify notochord-derived Shh ligand in the context of neural patterning, provides several new insights into mechanisms of Shh morphogen action.     

Regulation of BMP activity and range in Drosophila wing development

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

Dynamic changes in the response of cells to positive hedgehog signaling during mouse limb patterning

In the vertebrate limb, the posteriorly located zone of polarizing activity (ZPA) regulates digit identity through the morphogen Sonic Hedgehog (Shh). By genetically marking Shh-responding cells in mice, we have addressed whether the cumulative influence of positive Shh signaling over time and space reflects a linear gradient of Shh responsiveness and whether Shh could play additional roles in limb patterning. Our results show that all posterior limb mesenchyme cells, as well as the ectoderm, respond to Shh from the ZPA and become the bone, muscle, and skin of the posterior limb. Further, the readout of Shh activator function integrated over time and space does not display a stable and linear gradient along the A-P axis, as in a classical morphogen view. Finally, by fate mapping Shh-responding cells in Gli2 and Gli3 mutant limbs, we demonstrate that a specific level of positive Hh signaling is not required to specify digit identities.     

Sonic hedgehog regulates Gli activator and repressor functions with spatial and temporal precision in the mid/hindbrain region

The midbrain and anterior hindbrain offer an ideal system in which to study the coordination of tissue growth and patterning in three dimensions. Two organizers that control anteroposterior (AP) and dorsoventral (DV) development are known, and the regulation of AP patterning by Fgf8 has been studied in detail. Much less is known about the mechanisms that control mid/hindbrain development along the DV axis. Using a conditional mutagenesis approach, we have determined how the ventrally expressed morphogen sonic hedgehog (Shh) directs mid/hindbrain development over time and space through positive regulation of the Gli activators (GliA) and inhibition of the Gli3 repressor (Gli3R). We have discovered that Gli2A-mediated Shh signaling sequentially induces ventral neurons along the medial to lateral axis, and only before midgestation. Unlike in the spinal cord, Shh signaling plays a major role in patterning of dorsal structures (tectum and cerebellum). This function of Shh signaling involves inhibition of Gli3R and continues after midgestation. Gli3R levels also regulate overall growth of the mid/hindbrain region, and this largely involves the suppression of cell death. Furthermore, inhibition of Gli3R by Shh signaling is required to sustain expression of the AP organizer gene Fgf8. Thus, the precise spatial and temporal regulation of Gli2A and Gli3R by Shh is instrumental in coordinating mid/hindbrain development in three dimensions.     

Lipid modifications of Sonic hedgehog ligand dictate cellular reception and signal response

Background

Sonic hedgehog (Shh) signaling regulates cell growth during embryonic development, tissue homeostasis and tumorigenesis. Concentration-dependent cellular responses to secreted Shh protein are essential for tissue patterning. Shh ligand is covalently modified by two lipid moieties, cholesterol and palmitate, and their hydrophobic properties are known to govern the cellular release and formation of soluble multimeric Shh complexes. However, the influences of the lipid moieties on cellular reception and signal response are not well understood.

Methodology/Principal Findings

We analyzed fully lipidated Shh and mutant forms to eliminate one or both adducts in NIH3T3 mouse embryonic fibroblasts. Quantitative measurements of recombinant Shh protein concentration, cellular localization, and signaling potency were integrated to determine the contributions of each lipid adduct on ligand cellular localization and signaling potency. We demonstrate that lipid modification is required for cell reception, that either adduct is sufficient to confer cellular association, that the cholesterol adduct anchors ligand to the plasma membrane and that the palmitate adduct augments ligand internalization. We further show that signaling potency correlates directly with cellular concentration of Shh ligand.

Conclusions/Significance

The findings of this study demonstrate that lipid modification of Shh determines cell concentration and potency, revealing complementary functions of hydrophobic modification in morphogen signaling by attenuating cellular release and augmenting reception of Shh protein in target tissues.     

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.     

Pentagone: patrolling BMP morphogen signaling

Orchestration of spatial organization by signaling gradients--morphogen gradients--is a fundamental principle in animal development. Despite their importance in tissue patterning and growth, the exact mechanisms underlying the establishment and maintenance of morphogen gradients are poorly understood. Our recent work on BMP (bone morphogenetic protein) morphogen signaling during wing development identified a novel protein, Pentagone (Pent), as a critical regulator of morphogen activity. In the following, we discuss the properties of Pent and its role as a feed-back loop in morphogen gradient formation.     

Pentagone: patrolling BMP morphogen signaling

Orchestration of spatial organization by signaling gradients - morphogen gradients - is a fundamental principle in animal development. Despite their importance in tissue patterning and growth, the exact mechanisms underlying the establishment and maintenance of morphogen gradients are poorly understood. Our recent work on BMP (bone morphogenetic protein) morphogen signaling during wing development identified a novel protein, Pentagone (Pent), as a critical regulator of morphogen activity. In the following, we discuss the properties of Pent and its role as a feed-back loop in morphogen gradient formation.     

Region-specific requirement for cholesterol modification of sonic hedgehog in patterning the telencephalon and spinal cord

Sonic hedgehog (Shh) secreted from the axial signaling centers of the notochord and prechordal plate functions as a morphogen in dorsoventral patterning of the neural tube. Active Shh is uniquely cholesterol-modified and the hydrophobic nature of cholesterol suggests that it might regulate Shh spreading in the neural tube. Here, we examined the capacity of Shh lacking the cholesterol moiety (ShhN) to pattern different cell types in the telencephalon and spinal cord. In mice expressing ShhN, we detected low-level ShhN in the prechordal plate and notochord, consistent with the notion that ShhN can rapidly spread from its site of synthesis. Surprisingly, we found that low-level ShhN can elicit the generation of a full spectrum of ventral cell types in the spinal cord, whereas ventral neuronal specification and ganglionic eminence development in the Shh(N/-) telencephalon were severely impaired, suggesting that telencephalic patterning is more sensitive to alterations in local Shh concentration and spreading. In agreement, we observed induction of Shh pathway activity and expression of ventral markers at ectopic sites in the dorsal telencephalon indicative of long-range ShhN activity. Our findings indicate an essential role for the cholesterol moiety in restricting Shh dilution and deregulated spread for patterning the telencephalon. We propose that the differential effect of ShhN in patterning the spinal cord versus telencephalon may be attributed to regional differences in the maintenance of Shh expression in the ventral neuroepithelium and differences in dorsal tissue responsiveness to deregulated Shh spreading behavior.     

Disrupted dorsal neural tube BMP signaling in the cilia mutant Arl13b hnn stems from abnormal Shh signaling

In the embryonic neural tube, multiple signaling pathways work in concert to create functional neuronal circuits in the adult spinal cord. In the ventral neural tube, Sonic hedgehog (Shh) acts as a graded morphogen to specify neurons necessary for movement. In the dorsal neural tube, bone morphogenetic protein (BMP) and Wnt signals cooperate to specify neurons involved in sensation. Several signaling pathways, including Shh, rely on primary cilia in vertebrates. In this study, we used a mouse mutant with abnormal cilia, Arl13b(hnn), to study the relationship between cilia, cell signaling, and neural tube patterning. Arl13b(hnn) mutants have abnormal ventral neural tube patterning due to disrupted Shh signaling; in addition, dorsal patterning defects occur, but the cause of these is unknown. Here we show that the Arl13b(hnn) dorsal patterning defects result from abnormal BMP signaling. In addition, we find that Wnt ligands are abnormally expressed in Arl13b(hnn) mutants; surprisingly, however, downstream Wnt signaling is normal. We demonstrate that Arl13b is required non-autonomously for BMP signaling and Wnt ligand expression, indicating that the abnormal Shh signaling environment in Arl13b(hnn) embryos indirectly causes dorsal defects.     

Dynamic Assignment and Maintenance of Positional Identity in the Ventral Neural Tube by the Morphogen Sonic Hedgehog

Morphogens are secreted signalling molecules that act in a graded manner to control the pattern of cellular differentiation in developing tissues. An example is Sonic hedgehog (Shh), which acts in several developing vertebrate tissues, including the central nervous system, to provide positional information during embryonic patterning. Here we address how Shh signalling assigns the positional identities of distinct neuronal subtype progenitors throughout the ventral neural tube. Assays of intracellular signal transduction and gene expression indicate that the duration as well as level of signalling is critical for morphogen interpretation. Progenitors of the ventral neuronal subtypes are established sequentially, with progressively more ventral identities requiring correspondingly higher levels and longer periods of Shh signalling. Moreover, cells remain sensitive to changes in Shh signalling for an extended time, reverting to antecedent identities if signalling levels fall below a threshold. Thus, the duration of signalling is important not only for the assignment but also for the refinement and maintenance of positional identity. Together the data suggest a dynamic model for ventral neural tube patterning in which positional information corresponds to the time integral of Shh signalling. This suggests an alternative to conventional models of morphogen action that rely solely on the level of signalling.     

Pre-steady-state decoding of the Bicoid morphogen gradient

Morphogen gradients are established by the localized production and subsequent diffusion of signaling molecules. It is generally assumed that cell fates are induced only after morphogen profiles have reached their steady state. Yet, patterning processes during early development occur rapidly, and tissue patterning may precede the convergence of the gradient to its steady state. Here we consider the implications of pre-steady-state decoding of the Bicoid morphogen gradient for patterning of the anterior–posterior axis of the Drosophila embryo. Quantitative analysis of the shift in the expression domains of several Bicoid targets (gap genes) upon alteration of bcd dosage, as well as a temporal analysis of a reporter for Bicoid activity, suggest that a transient decoding mechanism is employed in this setting. We show that decoding the pre-steady-state morphogen profile can reduce patterning errors caused by fluctuations in the rate of morphogen production. This can explain the surprisingly small shifts in gap and pair-rule gene expression domains observed in response to alterations in bcd dosage.