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.     

An FGF autocrine loop initiated in second heart field mesoderm regulates morphogenesis at the arterial pole of the heart

In order to understand how secreted signals regulate complex morphogenetic events, it is crucial to identify their cellular targets. By conditional inactivation of Fgfr1 and Fgfr2 and overexpression of the FGF antagonist sprouty 2 in different cell types, we have dissected the role of FGF signaling during heart outflow tract development in mouse. Contrary to expectation, cardiac neural crest and endothelial cells are not primary paracrine targets. FGF signaling within second heart field mesoderm is required for remodeling of the outflow tract: when disrupted, outflow myocardium fails to produce extracellular matrix and TGFbeta and BMP signals essential for endothelial cell transformation and invasion of cardiac neural crest. We conclude that an autocrine regulatory loop, initiated by the reception of FGF signals by the mesoderm, regulates correct morphogenesis at the arterial pole of the heart. These findings provide new insight into how FGF signaling regulates context-dependent cellular responses during development.     

A Highlights from MBoC Selection: Synergistic interaction between the fibroblast growth factor and bone morphogenetic protein signaling pathways in lens cells

Fibroblast growth factors (FGFs) play a central role in two processes essential for lens transparency—fiber cell differentiation and gap junction–mediated intercellular communication (GJIC). Using serum-free primary cultures of chick lens epithelial cells (DCDMLs), we investigated how the FGF and bone morphogenetic protein (BMP) signaling pathways positively cooperate to regulate lens development and function. We found that culturing DCDMLs for 6 d with the BMP blocker noggin inhibits the canonical FGF-to-ERK pathway upstream of FRS2 activation and also prevents FGF from stimulating FRS2- and ERK-independent gene expression, indicating that BMP signaling is required at the level of FGF receptors. Other experiments revealed a second type of BMP/FGF interaction by which FGF promotes expression of BMP target genes as well as of BMP4. Together these studies reveal a novel mode of cooperation between the FGF and BMP pathways in which BMP keeps lens cells in an optimally FGF-responsive state and, reciprocally, FGF enhances BMP-mediated gene expression. This interaction provides a mechanistic explanation for why disruption of either FGF or BMP signaling in the lens leads to defects in lens development and function.     

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.     

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 BMP signaling gradient is interpreted through concentration thresholds in dorsal–ventral axial patterning

Bone Morphogenetic Protein (BMP) patterns the dorsal–ventral (DV) embryonic axis in all vertebrates, but it is unknown how cells along the DV axis interpret and translate the gradient of BMP signaling into differential gene activation that will give rise to distinct cell fates. To determine the mechanism of BMP morphogen interpretation in the zebrafish gastrula, we identified 57 genes that are directly activated by BMP signaling. By using Seurat analysis of single-cell RNA sequencing (scRNA-seq) data, we found that these genes are expressed in at least 3 distinct DV domains of the embryo. We distinguished between 3 models of BMP signal interpretation in which cells activate distinct gene expression through interpretation of thresholds of (1) the BMP signaling gradient slope; (2) the BMP signal duration; or (3) the level of BMP signal activation. We tested these 3 models using quantitative measurements of phosphorylated Smad5 (pSmad5) and by examining the spatial relationship between BMP signaling and activation of different target genes at single-cell resolution across the embryo. We found that BMP signaling gradient slope or BMP exposure duration did not account for the differential target gene expression domains. Instead, we show that cells respond to 3 distinct levels of BMP signaling activity to activate and position target gene expression. Together, we demonstrate that distinct pSmad5 threshold levels activate spatially distinct target genes to pattern the DV axis.

This study tested three models of how a BMP morphogen gradient is translated into differential gene activation that specifies distinct cell fates, finding that BMP signal concentration thresholds, not gradient shape or signal duration, position three distinct gene activation domains.     

Sox3 expression is maintained by FGF signaling and restricted to the neural plate by Vent proteins in the Xenopus embryo

The formation of the nervous system is initiated when ectodermal cells adopt the neural fate. Studies in Xenopus demonstrate that inhibition of BMP results in the formation of neural tissue. However, the molecular mechanism driving the expression of early neural genes in response to this inhibition is unknown. Moreover, controversy remains regarding the sufficiency of BMP inhibition for neural induction. To address these questions, we performed a detailed analysis of the regulation of the soxB1 gene, sox3, one of the earliest genes expressed in the neuroectoderm. Using ectodermal explant assays, we analyzed the role of BMP, Wnt and FGF signaling in the regulation of sox3 and the closely related soxB1 gene, sox2. Our results demonstrate that both sox3 and sox2 are induced in response to BMP antagonism, but by distinct mechanisms and that the activation of both genes is independent of FGF signaling. However, both require FGF for the maintenance of their expression. Finally, sox3 genomic elements were identified and characterized and an element required for BMP-mediated repression via Vent proteins was identified through the use of transgenesis and computational analysis. Interestingly, none of the elements required for sox3 expression were identified in the sox2 locus. Together our data indicate that two closely related genes have unique mechanisms of gene regulation at the onset of neural development.     

Patterning of mouse embryonic stem cell-derived pan-mesoderm by Activin A/Nodal and Bmp4 signaling requires Fibroblast Growth Factor activity

Abstract Embryonic stem (ES) cells have the potential to differentiate into all cell types of the adult body, and could allow regeneration of damaged tissues. The challenge is to alter differentiation toward functional cell types or tissues by directing ES cells to a specific fate. Efforts have been made to understand the molecular mechanisms that are required for the formation of the different germ layers and tissues from ES cells, and these mechanisms appear to be very similar in the mouse embryo. Differentiation toward mesoderm and mesoderm derivatives such as cardiac tissue or hemangioblasts has been demonstrated; however, the roles of Activin A/Nodal, bone morphogenetic protein (BMP), and fibroblast growth factor (FGF) signaling in the early patterning of ES cell-derived pan-mesoderm and anterior visceral endoderm (aVE) have not been reported yet. We therefore analyzed the roles of Activin A/Nodal, BMP, and FGF signaling in the patterning of ES cell-derived mesoderm as well as specification of the aVE by using a dual ES cell differentiation system combining a loss-of-function with a gain-of-function approach. We found that Activin A or Nodal directed the nascent mesoderm toward axial mesoderm and mesendoderm, while Bmp4 was inducing posterior and extraembryonic mesoderm at the expense of anterior primitive streak cells. FGF signaling appeared to have an important role in mesoderm differentiation by allowing an epithelial-to-mesenchymal transition of the newly formed mesoderm cells that would lead to their further patterning. Moreover, inhibition of FGF signaling resulted in increased expression of axial mesoderm markers. Additionally, we revealed that the formation of aVE cells from ES cells requires FGF-dependent Activin A/Nodal signaling and the attenuation of Bmp4 signaling.     

Spatial and temporal expression of heparan sulfate in mouse development regulates FGF and FGF receptor assembly

Heparan sulfate (HS) interacts with diverse growth factors, including Wnt, Hh, BMP, VEGF, EGF, and FGF family members, and is a necessary component for their signaling. These proteins regulate multiple cellular processes that are critical during development. However, a major question is whether developmental changes occur in HS that regulate the activity of these factors. Using a ligand and carbohydrate engagement assay, and focusing on FGF1 and FGF8b interactions with FGF receptor (FR)2c and FR3c, this paper reveals global changes in HS expression in mouse embryos during development that regulate FGF and FR complex assembly. Furthermore, distinct HS requirements are identified for both complex formation and signaling for each FGF and FR pair. Overall, these results suggest that changes in HS act as critical temporal regulators of growth factor and morphogen signaling during embryogenesis.     

FGF/MAPK signaling is required in the gastrula epiblast for avian neural crest induction

Neural crest induction involves the combinatorial inputs of the FGF, BMP and Wnt signaling pathways. Recently, a two-step model has emerged where BMP attenuation and Wnt activation induces the neural crest during gastrulation, whereas activation of both pathways maintains the population during neurulation. FGF is proposed to act indirectly during the inductive phase by activating Wnt ligand expression in the mesoderm. Here, we use the chick model to investigate the role of FGF signaling in the amniote neural crest for the first time and uncover a novel requirement for FGF/MAPK signaling. Contrary to current models, we demonstrate that FGF is required within the prospective neural crest epiblast during gastrulation and is unlikely to operate through mesodermal tissues. Additionally, we show that FGF/MAPK activity in the prospective neural plate prevents the ectopic expression of lateral ectoderm markers, independently of its role in neural specification. We then investigate the temporal participation of BMP/Smad signaling and suggest a later involvement in neural plate border development, likely due to widespread FGF/MAPK activity in the gastrula epiblast. Our results identify an early requirement for FGF/MAPK signaling in amniote neural crest induction and suggest an intriguing role for FGF-mediated Smad inhibition in ectodermal development.     

Cell Geometry Guides the Dynamic Targeting of Apoplastic GPI-Linked Lipid Transfer Protein to Cell Wall Elements and Cell Borders in Arabidopsis thaliana

During cellular morphogenesis, changes in cell shape and cell junction topology are fundamental to normal tissue and organ development. Here we show that apoplastic Glycophosphatidylinositol (GPI)-anchored Lipid Transfer Protein (LTPG) is excluded from cell junctions and flat wall regions, and passively accumulates around their borders in the epidermal cells of Arabidopsis thaliana. Beginning with intense accumulation beneath highly curved cell junction borders, this enrichment is gradually lost as cells become more bulbous during their differentiation. In fully mature epidermal cells, YFP-LTPG often shows a fibrous cellulose microfibril-like pattern within the bulging outer faces. Physical contact between a flat glass surface and bulbous cell surface induces rapid and reversible evacuation from contact sites and accumulation to the curved wall regions surrounding the contact borders. Thus, LTPG distribution is dynamic, responding to changes in cell shape and wall curvature during cell growth and differentiation. We hypothesize that this geometry-based mechanism guides wax-carrying LTPG to functional sites, where it may act to “seal” the vulnerable border surrounding cell-cell junctions and assist in cell wall fortification and cuticular wax deposition.     

Cdc2-like kinase 2 (Clk2) promotes early neural development in Xenopus embryos

Neural induction and patterning in vertebrates are regulated during early development by several morphogens, such as bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs). Ventral ectoderm differentiates into epidermis in response to BMPs, whereas BMP signaling is tightly inhibited in the dorsal ectoderm which develops into neural tissues. Here, we show that Cdc2-like kinase 2 (Clk2) promotes early neural development and inhibits epidermis differentiation in Xenopus embryos. clk2 is specifically expressed in neural tissues along the anterior-posterior axis during early Xenopus embryogenesis. When overexpressed in ectodermal explants, Clk2 induces the expression of both anterior and posterior neural marker genes. In agreement with this observation, overexpression of Clk2 in whole embryos expands the neural plate at the expense of epidermal ectoderm. Interestingly, the neural-inducing activity of Clk2 is increased following BMP inhibition and activation of the FGF signaling pathway in ectodermal explants. Clk2 also downregulates the level of p-Smad1/5/8 in cooperation with BMP inhibition, in addition to increasing the level of activated MAPK together with FGF. These results suggest that Clk2 plays a role in early neural development of Xenopus possibly via modulation of morphogen signals such as the BMP and FGF pathways.     

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.     

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.     

Inhibition of Transforming Growth Factor β (TGF-β) Signaling can Substitute for Oct4 Protein in Reprogramming and Maintain Pluripotency

Mouse pluripotent stem cells (PSCs), such as ES cells and induced PSCs (iPSCs), are an excellent system to investigate the molecular and cellular mechanisms involved in early embryonic development. The signaling pathways orchestrated by leukemia inhibitor factor/STAT3, Wnt/β-catenin, and FGF/MEK/ERK play key roles in the generation of pluripotency. However, the function of TGF-β signaling in this process remains elusive. Here we show that inhibiting TGF-β signaling with its inhibitor SB431542 can substitute for Oct4 during reprogramming. Moreover, inhibiting TGF-β signaling can sustain the pluripotency of iPSCs and ES cells through modulating FGF/MEK/ERK signaling. Therefore, this study reveals a novel function of TGF-β signaling inhibition in the generation and maintenance of PSCs.