Morphogen Gradients Research Articles

Differential compartmentalization of BMP4/NOGGIN requires NOGGIN trans-epithelial transport.

Using self-organizing human models of gastrulation, we previously showed that (1) BMP4 initiates the cascade of events leading to gastrulation, (2) BMP4 signal reception is restricted to the basolateral domain, and (3) in a human-specific manner, BMP4 directly induces the expression of NOGGIN. Here, we report the surprising discovery that in human epiblasts, NOGGIN and BMP4 were secreted into opposite extracellular spaces. Interestingly, apically presented NOGGIN could inhibit basally delivered BMP4. Apically imposed microfluidic flow demonstrated that NOGGIN traveled in the apical extracellular space. Our co-localization analysis detailed the endocytotic route that trafficked NOGGIN from the apical space to the basolateral intercellular space where BMP4 receptors were located. This apical-basal transcytosis was indispensable for NOGGIN inhibition. Taken together, the segregation of activator/inhibitor into distinct extracellular spaces challenges classical views of morphogen movement. We propose that the transport of morphogen inhibitors regulates the spatial availability of morphogens during embryogenesis.

Neural tube patterning: From a minimal model for rostrocaudal patterning toward an integrated 3D model

SummaryRostrocaudal patterning of the neural tube is a defining event in vertebrate brain development. This process is driven by morphogen gradients which specify the fate of neural progenitor cells, leading to the partitioning of the tube. Although this is extensively studied experimentally, an integrated view of the genetic circuitry is lacking. Here, we present a minimal gene regulatory model for rostrocaudal patterning, whose tristable topology was determined in a data-driven way. Using this model, we identified the repression of hindbrain fate as promising strategy for the improvement of current protocols for the generation of dopaminergic neurons. Furthermore, we combined our model with an established minimal model for dorsoventral patterning on a realistic 3D neural tube and found that key features of neural tube patterning could be recapitulated. Doing so, we demonstrate how data and models from different sources can be combined to simulate complex in vivo processes.

Getting Hedgehogs Where They Need to Go: Furin Cleavage Activates Dispatched for Sonic Hedgehog Transport

Hedgehog ligands activate an evolutionarily conserved signaling pathway that provides instructional cues during tissue morphogenesis, and when corrupted, contributes to developmental disorders and cancer. The transmembrane protein Dispatched is an essential component of the machinery that deploys Hedgehog family ligands from producing cells, and is required for Hedgehog morphogen gradient formation and signaling to long-range targets. Molecular mechanisms that regulate Dispatched to control Hedgehog release are not yet defined. We reveal that Dispatched is activated by Furin-mediated cleavage at a conserved processing site in its first extracellular loop. Disruption of Furin cleavage alters Dispatched membrane trafficking and attenuates Sonic Hedgehog release from ligand-producing cells. CRISPR/Cas9 gene editing was used to generate transgenic mice harboring a cleavage site mutation in the endogenous Dispatched locus. Homozygous animals show neuronal patterning phenotypes and decreased viability, confirming a requirement for Dispatched processing in vivo. As such, Furin serves as a crucial post-translational regulator of Dispatched activity in Hedgehog-producing cells during tissue morphogenesis.

Cell-Intrinsic WNT4 Promotes Hematopoietic Stem and Progenitor Cell Self-Renewal

Although intracellular Wnt signaling pathways need to be tightly regulated to promote hematopoietic stem cell self-renewal, the source and identity of important Wnt ligands in the bone marrow is still largely unknown. The noncanonical ligand Wnt4 is expressed in the bone marrow as well as in the stroma, and its overexpression in fetal liver cells facilitates thymic recovery; however, its impact on adult hematopoietic stem cell function remains unclear. Here, we report that the deletion of Wnt4 from hematopoietic cells in mice (Wnt4Δ/Δ ) resulted in decreased lymphopoiesis at steady state. This was likely at least in part due to the increased proinflammatory environment present in the bone marrow of Wnt4Δ/Δ mice. Wnt4Δ/Δ hematopoietic stem cells displayed reduced reconstitution capacity in serial transplants, thus demonstrating defective self-renewal, and they expanded poorly in response to lipopolysaccharide stimulation. This appeared to be the result of the absence of Wnt4 in stem/progenitor cells, as myeloid-restricted Wnt4 deletion had no notable effect. Finally, we observed that Wnt4Δ/Δ stem/progenitor cells were more quiescent, presenting enhanced levels of stress-associated JNK phosphorylation and p16INK4a expression, likely contributing to the reduced expansion observed in transplants. In conclusion, our results identify a new, largely autocrine role for Wnt4 in hematopoietic stem cell self-renewal, suggesting that regulation of Wnt signaling in hematopoiesis may not need Wnt secretion and could be independent of morphogen gradients.

Generation of extracellular morphogen gradients: the case for diffusion.

Cells within developing tissues rely on morphogens to assess positional information. Passive diffusion is the most parsimonious transport model for long-range morphogen gradient formation but does not, on its own, readily explain scaling, robustness and planar transport. Here, we argue that diffusion is sufficient to ensure robust morphogen gradient formation in a variety of tissues if the interactions between morphogens and their extracellular binders are considered. A current challenge is to assess how the affinity for extracellular binders, as well as other biophysical and cell biological parameters, determines gradient dynamics and shape in a diffusion-based transport system. Technological advances in genome editing, tissue engineering, live imaging and in vivo biophysics are now facilitating measurement of these parameters, paving the way for mathematical modelling and a quantitative understanding of morphogen gradient formation and modulation.

Patterning Neuroepithelial Cell Sheet via a Sustained Chemical Gradient Generated by Localized Passive Diffusion Devices.

Recent advances in human pluripotent stem cells (hPSCs)-derived in vitro models open a new avenue for studying early stage human development. While current approaches leverage the self-organizing capability of hPSCs, it remains unclear whether extrinsic morphogen gradients are sufficient to pattern neuroectoderm tissues in vitro. While microfluidics or hydrogel-based approaches to generate chemical gradients are well-established, these systems either require continuous pumping or encapsulating cells in gels, making it difficult for adaptation in standard biology laboratories and downstream analysis. In this work, we report a new device design that leverages localized passive diffusion, or LPaD for short, to generate a stable chemical gradient in an open environment. As LPaD is operated simply by media changing, common issues for microfluidic systems such as leakage, bubble formation, and contamination can be avoided. The device contains a slit carved in a film filled with solid gelatin and connected to a static aqueous morphogen reservoir. Concentration gradients generated by the device were visualized via DAPI fluorescent intensity and were found to be stable for up to 168 h. Using this device, we successfully induced cellular response of Madin-Darby canine kidney (MDCK) cells to the concentration gradient of a small-molecule drug, cytochalasin D. Furthermore, we efficiently patterned the dorsal-ventral axis of hPSC-derived forebrain neuroepithelial cells with the sonic hedgehog (Shh) signal gradient generated by the LPaD devices. Together, LPaD devices are powerful tools to control the local chemical microenvironment for engineering organotypic structures in vitro.

A Microfluidics Approach for the Functional Investigation of Signaling Oscillations Governing Somitogenesis.

Periodic segmentation of the presomitic mesoderm of a developing mouse embryo is controlled by a network of signaling pathways. Signaling oscillations and gradients are thought to control the timing and spacing of segment formation, respectively. While the involved signaling pathways have been studied extensively over the last decades, direct evidence for the function of signaling oscillations in controlling somitogenesis has been lacking. To enable the functional investigation of signaling dynamics, microfluidics is a previously established tool for the subtle modulation of these dynamics. With this microfluidics-based entrainment approach endogenous signaling oscillations are synchronized by pulses of pathway modulators. This enables modulation of, for instance, the oscillation period or the phase-relationship between two oscillating pathways. Furthermore, spatial gradients of pathway modulators can be established along the tissue to study how specific changes in the signaling gradients affect somitogenesis. The present protocol is meant to help establish microfluidic approaches for the first-time users of microfluidics. The basic principles and equipment needed to set up a microfluidic system are described, and a chip design is provided, with which a mold for chip generation can conveniently be prepared using a 3D printer. Finally, how to culture primary mouse tissue on a microfluidic chip and how to entrain signaling oscillations to external pulses of pathway modulators are discussed. This microfluidic system can also be adapted to harbor other in vivo and in vitro model systems such as gastruloids and organoids for functional investigation of signaling dynamics and morphogen gradients in other contexts.

Perlecan in Pericellular Mechanosensory Cell-Matrix Communication, Extracellular Matrix Stabilisation and Mechanoregulation of Load-Bearing Connective Tissues.

In this study, we review mechanoregulatory roles for perlecan in load-bearing connective tissues. Perlecan facilitates the co-acervation of tropoelastin and assembly of elastic microfibrils in translamellar cross-bridges which, together with fibrillin and elastin stabilise the extracellular matrix of the intervertebral disc annulus fibrosus. Pericellular perlecan interacts with collagen VI and XI to define and stabilize this matrix compartment which has a strategic position facilitating two-way cell-matrix communication between the cell and its wider extracellular matrix. Cues from the extracellular matrix are fed through this pericellular matrix back to the chondrocyte, allowing it to perceive and respond to subtle microenvironmental changes to regulate tissue homeostasis. Thus perlecan plays a key regulatory role in chondrocyte metabolism, and in chondrocyte differentiation. Perlecan acts as a transport proteoglycan carrying poorly soluble, lipid-modified proteins such as the Wnt or Hedgehog families facilitating the establishment of morphogen gradients that drive tissue morphogenesis. Cell surface perlecan on endothelial cells or osteocytes acts as a flow sensor in blood and the lacunar canalicular fluid providing feedback cues to smooth muscle cells regulating vascular tone and blood pressure, and the regulation of bone metabolism by osteocytes highlighting perlecan’s multifaceted roles in load-bearing connective tissues.

The GTPase Rab8 differentially controls the long- and short-range activity of the Hedgehog morphogen gradient by regulating Hedgehog apico-basal distribution.

The Hedgehog (Hh) morphogen gradient is required for patterning during metazoan development, yet the mechanisms involved in Hh apical and basolateral release and how this influences short- and long-range target induction are poorly understood. We found that depletion of the GTPase Rab8 in Hh-producing cells induces an imbalance between the level of apically and laterally released Hh. This leads to non-cell-autonomous differential effects on the expression of Hh target genes, namely an increase in its short-range targets and a concomitant decrease in long-range targets. We further found that Rab8 regulates the endocytosis and apico-basal distribution of Ihog, a transmembrane protein known to bind to Hh and to be crucial for establishment of the Hh gradient. Our data provide new insights into morphogen gradient formation, whereby morphogen activity is functionally distributed between apically and basolaterally secreted pools.

E3 ubiquitin ligase-mediated regulation of vertebrate ocular development; new insights into the function of SIAH enzymes.

Developmental regulation of the vertebrate visual system has been a focus of investigation for generations as understanding this critical time period has direct implications on our understanding of congenital blinding disease. The majority of studies to date have focused on transcriptional regulation mediated by morphogen gradients and signaling pathways. However, recent studies of post translational regulation during ocular development have shed light on the role of the ubiquitin proteasome system (UPS). This rather ubiquitous yet highly diverse system is well known for regulating protein function and localization as well as stability via targeting for degradation by the 26S proteasome. Work from many model organisms has recently identified UPS activity during various milestones of ocular development including retinal morphogenesis, retinal ganglion cell function as well as photoreceptor homeostasis. In particular work from flies and zebrafish has highlighted the role of the E3 ligase enzyme family, Seven in Absentia Homologue (Siah) during these events. In this review, we summarize the current understanding of UPS activity during Drosophila and vertebrate ocular development, with a major focus on recent findings correlating Siah E3 ligase activity with two major developmental stages of vertebrate ocular development, retinal morphogenesis and photoreceptor specification and survival.

Cytoneme delivery of Sonic Hedgehog from ligand-producing cells requires Myosin 10 and a Dispatched-BOC/CDON co-receptor complex.

Morphogens function in concentration-dependent manners to instruct cell fate during tissue patterning. The cytoneme morphogen transport model posits that specialized filopodia extend between morphogen-sending and responding cells to ensure that appropriate signaling thresholds are achieved. How morphogens are transported along and deployed from cytonemes, how quickly a cytoneme-delivered, receptor-dependent signal is initiated, and whether these processes are conserved across phyla are not known. Herein, we reveal that the actin motor Myosin 10 promotes vesicular transport of Sonic Hedgehog (SHH) morphogen in mouse cell cytonemes, and that SHH morphogen gradient organization is altered in neural tubes of Myo10-/- mice. We demonstrate that cytoneme-mediated deposition of SHH onto receiving cells induces a rapid, receptor-dependent signal response that occurs within seconds of ligand delivery. This activity is dependent upon a novel Dispatched (DISP)-BOC/CDON co-receptor complex that functions in ligand-producing cells to promote cytoneme occurrence and facilitate ligand delivery for signal activation.

Formation of a Morphogen Gradient - Acceleration by Quantum Walks

The establishment of graded concentration distributions of certain signalling molecules, called morphogens, is crucial for the successful embryonic development of multicellular organisms. The morphogen system that has been studied in the greatest detail is the Bicoid (Bcd) gradient established during the early embryonic development of fruit flies and provides the embryo cells with the necessary positional information for the proper implementation of the body plan of the organism. Both continuous as well as discrete-state classical random-walk models fail to provide a completely satisfactory explanation of the dynamics of Bcd gradient formation within the developmentally relevant timescales. By presuming the existence of an arbitrary chirality degree of freedom that characterizes each Bcd molecule, we here therefore develop a discrete-time quantum walk model of the process. Our approach is based on the recent suggestion that high temperatures and random interactions need not necessarily destroy many-body quantum coherence. In this way we are led to a system of non-classical master equations bearing an interference term that can be numerically solved on a computer. Our analysis predicts a large value of diffusion rate for the Bcd system in the very early nuclear division cycles that needs to be verified experimentally. Additionally, with the chirality and position degrees of freedom at our disposal entanglement shows up as a natural consequence in the system. Taking inspiration from a very recent demonstration of the existence of entanglement phenomenon in a hot, strongly-interacting atomic system, the discussion takes the point of view that the emergence of geometry in the embryo system is consequence of quantum entanglement that needs further understanding and analysis.

Spatiotemporal analysis of human intestinal development at single-cell resolution

SummaryDevelopment of the human intestine is not well understood. Here, we link single-cell RNA sequencing and spatial transcriptomics to characterize intestinal morphogenesis through time. We identify 101 cell states including epithelial and mesenchymal progenitor populations and programs linked to key morphogenetic milestones. We describe principles of crypt-villus axis formation; neural, vascular, mesenchymal morphogenesis, and immune population of the developing gut. We identify the differentiation hierarchies of developing fibroblast and myofibroblast subtypes and describe diverse functions for these including as vascular niche cells. We pinpoint the origins of Peyer’s patches and gut-associated lymphoid tissue (GALT) and describe location-specific immune programs. We use our resource to present an unbiased analysis of morphogen gradients that direct sequential waves of cellular differentiation and define cells and locations linked to rare developmental intestinal disorders. We compile a publicly available online resource, spatio-temporal analysis resource of fetal intestinal development (STAR-FINDer), to facilitate further work.

Epithelial colonies in vitro elongate through collective effects.

Epithelial tissues of the developing embryos elongate by different mechanisms, such as neighbor exchange, cell elongation, and oriented cell division. Since autonomous tissue self-organization is influenced by external cues such as morphogen gradients or neighboring tissues, it is difficult to distinguish intrinsic from directed tissue behavior. The mesoscopic processes leading to the different mechanisms remain elusive. Here, we study the spontaneous elongation behavior of spreading circular epithelial colonies in vitro. By quantifying deformation kinematics at multiple scales, we report that global elongation happens primarily due to cell elongations, and its direction correlates with the anisotropy of the average cell elongation. By imposing an external time-periodic stretch, the axis of this global symmetry breaking can be modified and elongation occurs primarily due to orientated neighbor exchange. These different behaviors are confirmed using a vertex model for collective cell behavior, providing a framework for understanding autonomous tissue elongation and its origins.

Minimal Synthetic Enhancers Reveal Control of the Probability of Transcriptional Engagement and its Timing by a Morphogen Gradient

Minimal Synthetic Enhancers Reveal Control of the Probability of Transcriptional Engagement and its Timing by a Morphogen Gradient

Strip it out and build it back! Engineering a morphogen gradient

Morphogens are biological molecules that instruct patterning in developing tissues. We used an artificial morphogen to guide development in the fruit fly wing. By doing so, we simplify the biological complexity, allowing us to decipher how morphogens work. - submission by Luca Cocconi, Marc de Gennes, Guillaume Salbreux

Morphometric and allometric rules of polyp's landscape in regular and chimeric coral colonies of the branching species Stylophora pistillata.

Most studies on architectural rules in corals have focused on the branch and the colony level, unveiling a variety of allometric rules. Working on the branching coral Stylophora pistillata, here we further extend the astogenic directives of this species at the polyp level, to reveal allometric and morphometric rules dictating polyps' arrangement. We identified a basic morphometric landscape as a six-polyp circlet developed around a founder polyp, with established distances between polyps (six equilateral triangles), reflecting a strong genetic-based background vs high plasticity on the population level. Testing these rules in regular and chimeric S. pistillata colonies, we revealed similar morphometric/allometric rules developed via a single astogenic pathway. In regular colonies, this pathway was driven by the presence/absence of intra-circlet budding polyps, while in chimeras, by the distances between the two founder polyps. In addition, we identified the intra-circlet budding as the origin of first branching, if BPC distances are kept <1.09 ± 0.25 mm. The emerged allometric/morphometric rules indicate the existence of a positional information paradigm for polyps' landscape distribution, where each polyp creates its own positional field of morphogen gradients through six inductive sites, thus forming six positional fields for the development of the archetypal "six-polyp crown".

Craniofacial and Long Bone Development in the Context of Distraction Osteogenesis.

Bone retains regenerative potential into adulthood, and surgeons harness this plasticity during distraction osteogenesis. The underlying biology governing bone development, repair, and regeneration is divergent between the craniofacial and appendicular skeleton. Each type of bone formation is characterized by unique molecular signaling and cellular behavior. Recent discoveries have elucidated the cellular and genetic processes underlying skeletal development and regeneration, providing an opportunity to couple biological and clinical knowledge to improve patient care. A comprehensive literature review of basic and clinical literature regarding craniofacial and long bone development, regeneration, and distraction osteogenesis was performed. The current understanding in craniofacial and long bone development and regeneration is discussed, and clinical considerations for the respective distraction osteogenesis procedures are presented. Distraction osteogenesis is a powerful tool to regenerate bone and thus address a number of craniofacial and appendicular skeletal deficiencies. The molecular mechanisms underlying bone regeneration, however, remain elusive. Recent work has determined that embryologic morphogen gradients constitute important signals during regeneration. In addition, striking discoveries have illuminated the cellular processes underlying mandibular regeneration during distraction osteogenesis, showing that skeletal stem cells reactivate embryologic neural crest transcriptomic processes to carry out bone formation during regeneration. Furthermore, innovative adjuvant therapies to complement distraction osteogenesis use biological processes active in embryogenesis and regeneration. Additional research is needed to further characterize the underlying cellular mechanisms responsible for improved bone formation through adjuvant therapies and the role skeletal stem cells play during regeneration.

Reconstitution of Morphogen Signaling Gradients in Cultured Cells.

Development of multicellular organisms depends on the proper establishment of signaling information in space and time. Secreted molecules called morphogens form concentration gradients in space and provide positional information to differentiating cells within the organism. Although the key molecular components of morphogen pathways have been identified, how the architectures and key parameters of morphogen pathways control the properties of signaling gradients, such as their size, speed, and robustness to perturbations, remains challenging to study in developing embryos. Reconstituting morphogen gradients in cell culture provides an alternative approach to address this question. Here we describe the methodology for reconstituting Sonic Hedgehog (SHH) signaling gradients in mouse fibroblast cells. The protocol includes the design of morphogen sending and receiving cell lines, the setup of radial and linear gradients, the quantitative time-lapse imaging, and the data analysis. Similar approaches could potentially be applied to other cell-cell communication pathways.

Neural Tube Patterning: From a Minimal Model for Rostrocaudal Patterning Towards an Integrated 3D Model

Rostrocaudal patterning of the neural tube is a key event in vertebrate brain development. Underlying this process are morphogen gradients which specify the fate of neural progenitor cells dose-dependently, leading to the partitioning of the tube. Although this is extensively studied experimentally, an integrated view of the genetic circuitry is currently lacking. Here, we present a minimal gene regulatory model for rostrocaudal patterning, whose configuration was determined in a data-driven way, resulting in a tristable network motif. Further analysis identified the repression of hindbrain fate as promising strategy for the improvement of current protocols for the generation of dopaminergic neurons. To test our model further, we integrated it with an established model for dorsoventral patterning to simulate the steady state pattern of a physiological 3D neural tube. Simulations of elevated morphogen secretion illustrate the sensitivity of neural tube patterning to morphogen levels, implying that these are tightly regulated during development.