Morphogen Distribution Research Articles

Ndst1,aheparan sulfatemodificationenzyme,regulates neuroectodermal patterningbyenhancingWntsignalingin Xenopus.

Neural tissue is derived from three precursor regions: neural plate, neural crest, and preplacodal ectoderm. These regions are determined by morphogen-mediated signaling. Morphogen distribution is generally regulated by binding to an extracellular matrix component, heparan sulfate (HS) proteoglycan. HS is modified by many enzymes, such as N-deacetyl sulfotransferase 1 (Ndst1), which is highly expressed in early development. However, functions of HS modifications in ectodermal patterning are largely unknown. In this study, we analyzed the role of Ndst1 using Xenopus embryos. We found that ndst1 was expressed in anterior neural plate and the trigeminal region at the neurula stage. ndst1 overexpression expanded the neural crest (NC) region, whereas translational inhibition reduced not only the trigeminal region, but also the adjacent NC region, especially the anterior part. At a later stage, ndst1 knocked-down embryos showed defects in cranial ganglion formation. We also found that Ndst1 activates Wnt signaling pathway at the neurula stage. Taken together, our results suggest that N-sulfonated HS accumulates Wnt ligand and activates Wnt signaling in ndst1-expressing cells, but that it inhibits signaling in non-ndst1-expressing cells, leading to proper neuroectodermal patterning.

Curvature in Biological Systems: Its Quantification, Emergence, and Implications across the Scales.

Surface curvature both emerges from, and influences the behavior of, living objects at length scales ranging from cellmembranes to single cells to tissues and organs. The relevance of surface curvature in biology is supported by numerous experimental and theoretical investigations in recent years. In this review, first, a brief introduction to the key ideas of surface curvature in the context of biological systems is given and the challenges that arise when measuring surface curvature are discussed. Giving an overview of the emergence of curvature in biological systems, its significance at different length scales becomes apparent. On the other hand, summarizing current findings also shows that both single cells and entire cell sheets, tissues or organisms respond to curvature by modulating their shape and their migration behavior. Finally, the interplay between the distribution of morphogens or micro-organisms and the emergence of curvature across length scales is addressed with examples demonstrating these key mechanistic principles of morphogenesis. Overall, this review highlights that curved interfaces are not merely a passive by-product of the chemical, biological, and mechanical processes but that curvature acts also as a signal that co-determines these processes.

The heparan sulfate modification enzyme, Hs6st1, governs Xenopus neuroectodermal patterning by regulating distributions of Fgf and Noggin

The nervous system has various types of cells derived from three neuroectodermal regions: neural plate (NP), neural crest (NC), and preplacodal ectoderm (PPE). Differentiation of these regions is regulated by various morphogens. However, regulatory mechanisms of morphogen distribution in neural patterning are still debated. In general, an extracellular component, heparan sulfate (HS), is essential to regulate morphogen gradients by modulating morphogen binding. The present study focused on an HS modification enzyme, heparan sulfate 6-O-sulfotransferase 1 (Hs6st1), which is highly expressed during the neurula stage in Xenopus. Our present in situ hybridization analysis revealed that Hs6st1 is expressed in the lateral sensorial layer of neuroectoderm. Overexpression of Hs6st1 expands Sox3 (NP marker gene) expression, and slightly dampens FoxD3 (NC marker) expression. Hs6st1 knockout using the CRISPR/Cas9 system also expands the neural plate region, followed by retinal malformation. These results imply that 6-O sulfation, mediated by Hs6st1, selectively regulates morphogen distribution required for neuroectodermal patterning. Among morphogens required for patterning, Fgf8a accumulates on Hs6st1-expressing cells, whereas a secreted BMP antagonist, Noggin, diffuses away from those cells. Thus, cell-autonomous 6-O sulfation of HS at the sensorial layer of neuroectoderm also affects neuroectodermal patterning in neighboring regions, including neural plate and neural crest, not only through accumulation, but also through dispersal of specific morphogens.

Determining the role of advection in patterning by bone morphogenetic proteins through neural network model-based acceleration of a 3D finite element model of the zebrafish embryo

Embryonic development is a complex phenomenon that integrates genetic regulation and biomechanical cellular behaviors. However, the relative influence of these factors on spatiotemporal morphogen distributions is not well understood. Bone Morphogenetic Proteins (BMPs) are the primary morphogens guiding the dorsal-ventral (DV) patterning of the early zebrafish embryo, and BMP signaling is regulated by a network of extracellular and intracellular factors that impact the range and signaling of BMP ligands. Recent advances in understanding the mechanism of pattern formation support a source-sink mechanism, however, it is not clear how the source-sink mechanism shapes the morphogen patterns in three-dimensional (3D) space, nor how sensitive the pattern is to biophysical rates and boundary conditions along both the anteroposterior (AP) and DV axes of the embryo, nor how the patterns are controlled over time. Throughout blastulation and gastrulation, major cell movement, known as epiboly, happens along with the BMP-mediated DV patterning. The layer of epithelial cells begins to thin as they spread toward the vegetal pole of the embryo until it has completely engulfed the yolk cell. This dynamic domain may influence the distributions of BMP network members through advection. We developed a Finite Element Model (FEM) that incorporates all stages of zebrafish embryonic development data and solves the advection-diffusion-reaction Partial Differential Equations (PDE) in a growing domain. We use the model to investigate mechanisms in underlying BMP-driven DV patterning during epiboly. Solving the PDE is computationally expensive for parameter exploration. To overcome this obstacle, we developed a Neural Network (NN) metamodel of the 3D embryo that is accurate and fast and provided a nonlinear map between high-dimensional input and output that replaces the direct numerical simulation of the PDEs. From the modeling and acceleration by the NN metamodels, we identified the impact of advection on patterning and the influence of the dynamic expression level of regulators on the BMP signaling network.

The nested embryonic dorsal domains of BMP-target genes are not scaled to size during the evolution of Drosophilaspecies.

Egg size is a fast-evolving trait among Drosophilids expected to change the spatial distribution of morphogens that pattern the embryonic axes. Here we asked whether the patterning of the dorsal region of the embryo by the Decapentaplegic/Bone Morphogenetic Protein-4 (DPP/BMP-4) gradient is scaled among Drosophila species with different egg sizes. This region specifies the extra-embryonic tissue amnioserosa and the ectoderm. We find that the entire dorsal region scales with embryo size, but the gene expression patterns regulated by DPP are not proportional, suggesting that the DPP gradient is differentially scaled during evolution. To further test whether the DPP gradient can scale or not in Drosophilamelanogaster, we created embryos with expanded dorsal regions that mimic changes in scale seen in other species and measured the resulting domains of DPP-target genes. We find that the proportions of these domains are not maintained, suggesting that the DPP gradient is unable to scale in the embryo. These and previous findings suggest that the embryonic dorso-ventral patterning lack scaling in the ventral and dorsal sides but is robust in the lateral region where the neuroectoderm is specified and two opposing gradients, Dorsal/NFkappa-B and DPP, intersect. We propose that the lack of scaling of the DPP gradient may contribute to changes in the size of the amnioserosa and the numbers of ectodermal cells with specific cortical tensions, which are expected to generate distinct mechanical forces for gastrulating embryos of different sizes.

Size matters: tissue size as a marker for a transition between reaction-diffusion regimes in spatio-temporal distribution of morphogens.

The reaction–diffusion model constitutes one of the most influential mathematical models to study distribution of morphogens in tissues. Despite its widespread use, the effect of finite tissue size on model-predicted spatio-temporal morphogen distributions has not been completely elucidated. In this study, we analytically investigated the spatio-temporal distributions of morphogens predicted by a reaction–diffusion model in a finite one-dimensional domain, as a proxy for a biological tissue, and compared it with the solution of the infinite-domain model. We explored the reduced parameter, the tissue length in units of a characteristic reaction–diffusion length, and identified two reaction–diffusion regimes separated by a crossover tissue size estimated in approximately three characteristic reaction–diffusion lengths. While above this crossover the infinite-domain model constitutes a good approximation, it breaks below this crossover, whereas the finite-domain model faithfully describes the entire parameter space. We evaluated whether the infinite-domain model renders accurate estimations of diffusion coefficients when fitted to finite spatial profiles, a procedure typically followed in fluorescence recovery after photobleaching (FRAP) experiments. We found that the infinite-domain model overestimates diffusion coefficients when the domain is smaller than the crossover tissue size. Thus, the crossover tissue size may be instrumental in selecting the suitable reaction–diffusion model to study tissue morphogenesis.

Hedgehog on track: Long-distant signal transport and transfer through direct cell-to-cell contact.

The function of Hedgehog (Hh) as a morphogen results from its long-distance distribution from producing to neighboring receiving cells within the developing tissue. This signal distribution enables, for example, the formation of a concentration gradient eliciting distinct cellular responses that will give rise to spatial patterning. Hh is a lipid modified protein and its dispersion is better guaranteed through cytonemes, cell protrusions that allow direct cell membrane contact and signal transfer at a distance. Hh and its receptor Patched (Ptc) meet at cytoneme contacts in a way that reminds synapses. Both Hh and Ptc require a recycling process prior to presentation in cytonemes. Increasing research on the role of cytonemes in Hh signaling is revealing cellular mechanisms that link signal transport through dynamic cytonemes with concurrent regulation of cell adhesion. The equilibrium between these two processes is being unveiled as crucial to both patterned morphogen distribution and signal transfer. In addition, these discoveries are pushing forward our understanding of the role of extracellular elements involved in the Hh pathway, such as the Hh coreceptors Ihog and Boi and the glypicans Dally and Dally-like protein (Dlp).

Glypican 4 mediates Wnt transport between germ layers via signaling filopodia.

Glypicans influence signaling pathways by regulating morphogen trafficking and reception. However, the underlying mechanisms in vertebrates are poorly understood. In zebrafish, Glypican 4 (Gpc4) is required for convergence and extension (C&E) of both the mesoderm and endoderm. Here, we show that transgenic expression of GFP-Gpc4 in the endoderm of gpc4 mutants rescued C&E defects in all germ layers. The rescue of mesoderm was likely mediated by Wnt5b and Wnt11f2 and depended on signaling filopodia rather than on cleavage of the Gpc4 GPI anchor. Gpc4 bound both Wnt5b and Wnt11f2 and regulated formation of the filopodia that transport Wnt5b and Wnt11f2 to neighboring cells. Moreover, this rescue was suppressed by blocking signaling filopodia that extend from endodermal cells. Thus, GFP-Gpc4-labeled protrusions that emanated from endodermal cells transported Wnt5b and Wnt11f2 to other germ layers, rescuing the C&E defects caused by a gpc4 deficiency. Our study reveals a new mechanism that could explain in vivo morphogen distribution involving Gpc4.

Improving the understanding of cytoneme-mediated morphogen gradients by in silico modeling.

Morphogen gradients are crucial for the development of organisms. The biochemical properties of many morphogens prevent their extracellular free diffusion, indicating the need of an active mechanism for transport. The involvement of filopodial structures (cytonemes) has been proposed for morphogen signaling. Here, we describe an in silico model based on the main general features of cytoneme-meditated gradient formation and its implementation into Cytomorph, an open software tool. We have tested the spatial and temporal adaptability of our model quantifying Hedgehog (Hh) gradient formation in two Drosophila tissues. Cytomorph is able to reproduce the gradient and explain the different scaling between the two epithelia. After experimental validation, we studied the predicted impact of a range of features such as length, size, density, dynamics and contact behavior of cytonemes on Hh morphogen distribution. Our results illustrate Cytomorph as an adaptive tool to test different morphogen gradients and to generate hypotheses that are difficult to study experimentally.

In Pursuit of Designing Multicellular Engineered Living Systems: A Fluid Mechanical Perspective

From intracellular protein signaling to embryonic symmetry-breaking, fluid transport ubiquitously drives biological events in living systems. We provide an overview of the fundamental fluid mechanics and transport phenomena across a range of length scales in cellular systems, with emphasis on how cellular functions are influenced by fluid transport. We also highlight how understanding the physical basis of these fluid dynamic phenomena can be implemented to engineer increasingly complex multicellular systems that recapitulate tissue-level functions. Examples discussed include the manipulation of intracellular fluid volume to achieve cell differentiation/dedifferentiation and the use of microfluidic systems to control the spatial and temporal distribution of morphogens and fluid forces to generate vascularized organoids.

Global shape of Toll activation is determined by wntD enhancer properties

Buffering variability in morphogen distribution is essential for reproducible patterning. A theoretically proposed class of mechanisms, termed "distal pinning," achieves robustness by combining local sensing of morphogen levels with global modulation of gradient spread. Here, we demonstrate a critical role for morphogen sensing by a gene enhancer, which ultimately determines the final global distribution of the morphogen and enables reproducible patterning. Specifically, we show that, while the pattern of Toll activation in the early Drosophila embryo is robust to gene dosage of its locally produced regulator, WntD, it is sensitive to a single-nucleotide change in the wntD enhancer. Thus, enhancer properties of locally produced WntD directly impinge on the global morphogen profile.

Impulsive signaling model of cytoneme-based morphogen gradient formation

Morphogen protein gradients play a vital role in regulating spatial pattern formation during development. The most commonly accepted mechanism of protein gradient formation involves the diffusion and degradation of morphogens from a localized source. However, there is growing experimental evidence for a direct cell-to-cell signaling mechanism via thin actin-rich cellular extensions known as cytonemes. Recent modeling studies of cytoneme-based morphogenesis in invertebrates ignore the discrete nature of vesicular transport along cytonemes, focusing on deterministic continuum models. In this paper, we develop an impulsive signaling model of morphogen gradient formation in invertebrates, which takes into account the discrete and stochastic nature of vesicular transport along cytonemes. We begin by solving a first passage time problem with sticky boundaries to determine the expected time to deliver a vesicle to a target cell, assuming that there is a ‘nucleation’ time for injecting the vesicle into the cytoneme. We then use queuing theory to analyze the impulsive model of morphogen gradient formation in the case of multiple cytonemes and multiple targets. In particular, we determine the steady-state mean and variance of the morphogen distribution across a one-dimensional array of target cells. The mean distribution recovers the spatially decaying morphogen gradient of previous deterministic models. However, the burst-like nature of morphogen transport can lead to Fano factors greater than unity across the array of cells, resulting in significant fluctuations at more distant target sites.

Gene expression analysis of potential morphogen signalling modifying factors in Panarthropoda

BackgroundMorphogen signalling represents a key mechanism of developmental processes during animal development. Previously, several evolutionary conserved morphogen signalling pathways have been identified, and their players such as the morphogen receptors, morphogen modulating factors (MMFs) and the morphogens themselves have been studied. MMFs are factors that regulate morphogen distribution and activity. The interactions of MMFs with different morphogen signalling pathways such as Wnt signalling, Hedgehog (Hh) signalling and Decapentaplegic (Dpp) signalling are complex because some of the MMFs have been shown to interact with more than one signalling pathway, and depending on genetic context, to have different, biphasic or even opposing function. This complicates the interpretation of expression data and functional data of MMFs and may be one reason why data on MMFs in other arthropods than Drosophila are scarce or totally lacking.ResultsAs a first step to a better understanding of the potential roles of MMFs in arthropod development, we investigate here the embryonic expression patterns of division abnormally delayed (dally), dally-like protein (dlp), shifted (shf) and secreted frizzled-related protein 125 (sFRP125) and sFRP34 in the beetle Tribolium castaneum, the spider Parasteatoda tepidariorum, the millipede Glomeris marginata and the onychophoran Euperipatoides kanangrensis. This pioneer study represents the first comprehensive comparative data set of these genes in panarthropods.ConclusionsExpression profiles reveal a high degree of diversity, suggesting that MMFs may represent highly evolvable nodes in otherwise conserved gene regulatory networks. Conserved aspects of MMF expression, however, appear to concern function in segmentation and limb development, two of the key topics of evolutionary developmental research.

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.

FRAP Analysis of Extracellular Diffusion in Zebrafish Embryos.

Morphogens are signaling molecules that provide positional information to cells during development. They must move through embryonic tissues in order to coordinate patterning. The rate of a morphogen's movement through a tissue-its effective diffusivity-affects the morphogen's distribution and therefore influences patterning. Fluorescence recovery after photobleaching (FRAP) is a powerful method to measure the effective diffusion of molecules through cells and tissues, and has been successfully employed to examine morphogen mobility and gain important insights into embryogenesis. Here, we provide detailed protocols for FRAP assays in vitro and in living zebrafish embryos, and weexplain how to analyze FRAP data using the open-source software PyFRAP to determine effective diffusion coefficients.

Stem Cell Extracellular Vesicles: Extended Messages of Regeneration.

Stem cells are critical to maintaining steady-state organ homeostasis and regenerating injured tissues. Recent intriguing reports implicate extracellular vesicles (EVs) as carriers for the distribution of morphogens and growth and differentiation factors from tissue parenchymal cells to stem cells, and conversely, stem cell–derived EVs carrying certain proteins and nucleic acids can support healing of injured tissues. We describe approaches to make use of engineered EVs as technology platforms in therapeutics and diagnostics in the context of stem cells. For some regenerative therapies, natural and engineered EVs from stem cells may be superior to single-molecule drugs, biologics, whole cells, and synthetic liposome or nanoparticle formulations because of the ease of bioengineering with multiple factors while retaining superior biocompatibility and biostability and posing fewer risks for abnormal differentiation or neoplastic transformation. Finally, we provide an overview of current challenges and future directions of EVs as potential therapeutic alternatives to cells for clinical applications.

3D Simulations of Morphogen Transport in an Early Fish Embryo

During the early stages of embryonic development, various means of cell communication orchestrate tissue development within the highly dynamic environment. Signalling gradients of morphogens determine cell fates, tissue generation, and in the long run organs of the final organism. In our research we focus on the distribution and effects of Wnt8a, a morphogen involved in the development and differentiation of the brain. In a previous work we were able to show experimentally that this protein is distributed by a novel short-range propagation mechanism by means of specialized filopodia[1]. Here we present our results on extending these simulations of a simple flat tissue towards a more accurate description of the embryo in a 3D environment.The simulation models the tissue expansion on a 3D spherical surface and morphogen distribution via filopodia formation. It integrates length and angle distributions as well as growth frequencies of the filopodia, cell migration, and a slight ligand decay consistently with experimental measurements. Additionally it extends our previous model by explicitly considering the movement of the Wnt8a source.[1] Filopodia-based Wnt transport during vertebrate tissue patterning. Stanganello, E., Hagemann, A. I. H., Mattes, B., Sinner, C., Meyen, D., Weber, S., Schug, A., Raz, E. and Scholpp, S., Nature Communications 6, 2015

The presence of nuclear cactus in the early Drosophila embryo may extend the dynamic range of the dorsal gradient.

In a developing embryo, the spatial distribution of a signaling molecule, or a morphogen gradient, has been hypothesized to carry positional information to pattern tissues. Recent measurements of morphogen distribution have allowed us to subject this hypothesis to rigorous physical testing. In the early Drosophila embryo, measurements of the morphogen Dorsal, which is a transcription factor responsible for initiating the earliest zygotic patterns along the dorsal-ventral axis, have revealed a gradient that is too narrow to pattern the entire axis. In this study, we use a mathematical model of Dorsal dynamics, fit to experimental data, to determine the ability of the Dorsal gradient to regulate gene expression across the entire dorsal-ventral axis. We found that two assumptions are required for the model to match experimental data in both Dorsal distribution and gene expression patterns. First, we assume that Cactus, an inhibitor that binds to Dorsal and prevents it from entering the nuclei, must itself be present in the nuclei. And second, we assume that fluorescence measurements of Dorsal reflect both free Dorsal and Cactus-bound Dorsal. Our model explains the dynamic behavior of the Dorsal gradient at lateral and dorsal positions of the embryo, the ability of Dorsal to regulate gene expression across the entire dorsal-ventral axis, and the robustness of gene expression to stochastic effects. Our results have a general implication for interpreting fluorescence-based measurements of signaling molecules.

Exosomes as Hedgehog carriers in cytoneme-mediated transport and secretion.

The Hedgehog signalling pathway is crucial for development, adult stem cell maintenance, cell migration and axon guidance in a wide range of organisms. During development, the Hh morphogen directs tissue patterning according to a concentration gradient. Lipid modifications on Hh are needed to achieve graded distribution, leading to debate about how Hh is transported to target cells despite being membrane-tethered. Cytonemes in the region of Hh signalling have been shown to be essential for gradient formation, but the carrier of the morphogen is yet to be defined. Here we show that Hh and its co-receptor Ihog are in exovesicles transported via cytonemes. These exovesicles present protein markers and other features of exosomes. Moreover, the cell machinery for exosome formation is necessary for normal Hh secretion and graded signalling. We propose Hh transport via exosomes along cytonemes as a significant mechanism for the restricted distribution of a lipid-modified morphogen.

Carrier of Wingless (Cow), a secreted heparan sulfate proteoglycan, promotes extracellular transport of Wingless.

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