Morphogen Gradients Research Articles

Mechano-gradients drive morphogen-noise correction to ensure robust patterning.

Morphogen gradients instruct cells to pattern tissues. Although the mechanisms by which morphogens transduce chemical signals have been extensively studied, the roles and regulation of the physical communication between morphogen-receiver cells remain unclear. Here, we show that the Wnt/β-catenin-morphogen gradient, which patterns the embryonic anterior-posterior (AP) axis, generates intercellular tension gradients along the AP axis by controlling membrane cadherin levels in zebrafish embryos. This "mechano-gradient" is used for the cell competition-driven correction of noisy morphogen gradients. Naturally and artificially generated unfit cells, producing noisy Wnt/β-catenin gradients, induce local deformation of the mechano-gradients that activate mechanosensitive calcium channels in the neighboring fit cells, which then secrete annexin A1a to kill unfit cells. Thus, chemo-mechanical interconversion-mediated competitive communication between the morphogen-receiver cells ensures precise tissue patterning.

Spatiotemporally programmed release of aptamer tethered dual angiogenic growth factors.

In tissue extracellular matrix (ECM), multiple growth factors (GFs) are sequestered through affinity interactions and released as needed by proteases, establishing spatial morphogen gradients in a time-controlled manner to guide cell behavior. Inspired by these ECM characteristics, we developed an "intelligent" biomaterial platform that spatially controls the combined bioavailability of multiple angiogenic GFs, specifically vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF-BB). Utilizing aptamer affinity interactions and complementary sequences within a GelMA matrix, our platform achieves on-demand, triggered release of individual GFs which can be "programmed" in temporally-controlled, repeatable cycles. The platform features stable incorporation of dual aptamers specific for both GFs, functional aptamer-CS molecular recognition in a 3D microenvironment with long-term stability of at least 15 days at physiological temperature, and spatially localized sequestration of individual GFs. Additionally, the system allows differential amounts of GFs to be released from the same hydrogels at different time-points, mimicking dynamic GF presentation in a 3D matrix similar to the native ECM. This flexible control over individual GF release kinetics opens new possibilities for dynamic GF presentation, with adjustable release profiles to meet the spatiotemporal needs of growing engineered tissue.

Confinement Modulates Axial Patterning in Regenerating Hydra

The establishment of the body plan is a major step in animal morphogenesis. The role of mechanical forces and feedback in patterning the body plan remains unclear. Here we explore this question by studying regenerating Hydra tissues confined in narrow cylindrical channels, which constrain their morphology. We find that frustration between the orientation of the channel and the inherited axis in the regenerating tissues can lead to the formation of a multiaxial body plan. The morphological outcome is directly related to the pattern of nematic topological defects that emerges in the organization of the supracellular actomyosin fibers. When the inherited axis, which can be read out from the initial alignment of the supracellular fibers in the confined spheroid, is parallel to the channel's axis, the tissue regenerates normally into animals with a single body axis aligned with the channel. However, regenerating spheroids that are confined in a frustrated perpendicular configuration often develop excess defects (including negatively charged −1/2 defects) and regenerate into multiaxial morphologies. The influence of mechanical constraints on the regenerated body plan argues against an axial patterning mechanism that is based solely on inherited gradients of biochemical morphogens. We further show that the dependence of the regeneration outcomes on the initial tissue orientation can be recapitulated by a biophysical model, which considers the coupled dynamics of the nematic organization of the actomyosin fibers and a morphogen concentration field, incorporating a mechanochemical feedback loop involving strain-dependent morphogen production at defect sites. Published by the American Physical Society 2024

Selective utilization of glucose metabolism guides mammalian gastrulation

The prevailing dogma for morphological patterning in developing organisms argues that the combined inputs of transcription factor networks and signalling morphogens alone generate spatially and temporally distinct expression patterns. However, metabolism has also emerged as a critical developmental regulator1–10, independent of its functions in energy production and growth. The mechanistic role of nutrient utilization in instructing cellular programmes to shape the in vivo developing mammalian embryo remains unknown. Here we reveal two spatially resolved, cell-type- and stage-specific waves of glucose metabolism during mammalian gastrulation by using single-cell-resolution quantitative imaging of developing mouse embryos, stem cell models and embryo-derived tissue explants. We identify that the first spatiotemporal wave of glucose metabolism occurs through the hexosamine biosynthetic pathway to drive fate acquisition in the epiblast, and the second wave uses glycolysis to guide mesoderm migration and lateral expansion. Furthermore, we demonstrate that glucose exerts its influence on these developmental processes through cellular signalling pathways, with distinct mechanisms connecting glucose with the ERK activity in each wave. Our findings underscore that—in synergy with genetic mechanisms and morphogenic gradients—compartmentalized cellular metabolism is integral in guiding cell fate and specialized functions during development. This study challenges the view of the generic and housekeeping nature of cellular metabolism, offering valuable insights into its roles in various developmental contexts.

Dynamics of morphogen source formation in a growing tissue.

A tight regulation of morphogen production is key for morphogen gradient formation and thereby for reproducible and organised organ development. Although many genetic interactions involved in the establishment of morphogen production domains are known, the biophysical mechanisms of morphogen source formation are poorly understood. Here we addressed this by focusing on the morphogen Sonic hedgehog (Shh) in the vertebrate neural tube. Shh is produced by the adjacently located notochord and by the floor plate of the neural tube. Using a data-constrained computational screen, we identified different possible mechanisms by which floor plate formation can occur, only one of which is consistent with experimental data. In this mechanism, the floor plate is established rapidly in response to Shh from the notochord and the dynamics of regulatory interactions within the neural tube. In this process, uniform activators and Shh-dependent repressors are key for establishing the floor plate size. Subsequently, the floor plate becomes insensitive to Shh and increases in size due to tissue growth, leading to scaling of the floor plate with neural tube size. In turn, this results in scaling of the Shh amplitude with tissue growth. Thus, this mechanism ensures a separation of time scales in floor plate formation, so that the floor plate domain becomes growth-dependent after an initial rapid establishment phase. Our study raises the possibility that the time scale separation between specification and growth might be a common strategy for scaling the morphogen gradient amplitude in growing organs. The model that we developed provides a new opportunity for quantitative studies of morphogen source formation in growing tissues.

Mobile signals, patterning, and positional information in root development.

Multicellular organisms use mobile intercellular signals to generate spatiotemporal patterns of growth and differentiation. These signals, termed morphogens, arise from localized sources and move by diffusion or directional transport to be interpreted at target cells. The classical model for a morphogen is where a substance diffuses from a source to generate a concentration gradient that provides positional information across a field. This concept, presented by Wolpert and popularized as the "French Flag Model," remains highly influential, but other patterning models, which do not rely on morphogen gradients, also exist. Here, we review current evidence for mobile morphogenetic signals in plant root development and how they fit within existing conceptual frameworks for pattern formation. We discuss how the signals are formed, distributed, and interpreted in space and time, emphasizing the regulation of movement on the ability of morphogens to specify patterns. While significant advances have been made in the field since the first identification of mobile morphogenetic factors in plants, key questions remain to be answered, such as how morphogen movement is regulated, how these mechanisms allow scaling in different species, and how morphogens act to enable plant regeneration in response to damage.

Robust tissue pattern formation by coupling morphogen signal and cell adhesion

Morphogens, locally produced signaling molecules, form a concentration gradient to guide tissue patterning. Tissue patterns emerge as a collaboration between morphogen diffusion and responsive cell behaviors, but the mechanisms through which diffusing morphogens define precise spatial patterns amidst biological fluctuations remain unclear. To investigate how cells respond to diffusing proteins to generate tissue patterns, we develop SYMPLE3D, a 3D culture platform. By engineering gene expression responsive to artificial morphogens, we observe that coupling morphogen signals with cadherin-based adhesion is sufficient to convert a morphogen gradient into distinct tissue domains. Morphogen-induced cadherins gather activated cells into a single domain, removing ectopically activated cells. In addition, we reveal a switch-like induction of cadherin-mediated compaction and cell mixing, homogenizing activated cells within the morphogen gradient to form a uniformly activated domain with a sharp boundary. These findings highlight the cooperation between morphogen gradients and cell adhesion in robust tissue patterning and introduce a novel method for tissue engineering to develop new tissue domains in organoids.

A polarized FGF8 source specifies frontotemporal signatures in spatially oriented cell populations of cortical assembloids

Organoids generating major cortical cell types in distinct compartments are used to study cortical development, evolution and disorders. However, the lack of morphogen gradients imparting cortical positional information and topography in current systems hinders the investigation of complex phenotypes. Here, we engineer human cortical assembloids by fusing an organizer-like structure expressing fibroblast growth factor 8 (FGF8) with an elongated organoid to enable the controlled modulation of FGF8 signaling along the longitudinal organoid axis. These polarized cortical assembloids mount a position-dependent transcriptional program that in part matches the in vivo rostrocaudal gene expression patterns and that is lost upon mutation in the FGFR3 gene associated with temporal lobe malformations and intellectual disability. By producing spatially oriented cell populations with signatures related to frontal and temporal area identity within individual assembloids, this model recapitulates in part the early transcriptional divergence embedded in the protomap and enables the study of cortical area-relevant alterations underlying human disorders.

DNA microbeads for spatio-temporally controlled morphogen release within organoids

AbstractOrganoids are transformative in vitro model systems that mimic features of the corresponding tissue in vivo. However, across tissue types and species, organoids still often fail to reach full maturity and function because biochemical cues cannot be provided from within the organoid to guide their development. Here we introduce nanoengineered DNA microbeads with tissue mimetic tunable stiffness for implementing spatio-temporally controlled morphogen gradients inside of organoids at any point in their development. Using medaka retinal organoids and early embryos, we show that DNA microbeads can be integrated into embryos and organoids by microinjection and erased in a non-invasive manner with light. Coupling a recombinant surrogate Wnt to the DNA microbeads, we demonstrate the spatio-temporally controlled morphogen release from the microinjection site, which leads to morphogen gradients resulting in the formation of retinal pigmented epithelium while maintaining neuroretinal cell types. Thus, we bioengineered retinal organoids to more closely mirror the cell type diversity of in vivo retinae. Owing to the facile, one-pot fabrication process, the DNA microbead technology can be adapted to other organoid systems for improved tissue mimicry.

How the zebra got its stripes: Curvature-dependent diffusion orients Turing patterns on three-dimensional surfaces.

Many animals have patterned fur, feathers, or scales, such as the stripes of a zebra. Turing models, or reaction-diffusion systems, are a class of mathematical models of interacting species that have been successfully used to generate animal-like patterns for many species. When diffusion of the inhibitor is high enough relative to the activator, a diffusion-driven instability can spontaneously form patterns. However, it is not just the type of pattern but also the orientation that matters, and it remains unclear how patterns are oriented in practice. Here, we propose a mechanism by which the curvature of the surface influences the rate of diffusion, and can recapture the correct orientation of stripes on models of a zebra and of a cat in numerical simulations. Previous work has shown how anisotropic diffusion can give stripe forming reaction-diffusion systems a bias in orientation. From the observation that zebra stripes run around the direction of highest curvature, that is around the torso and legs, we apply this result by modifying the anisotropic diffusion rates based on the local curvature. These results show how local geometry can influence the reaction dynamics to give robust, global-scale patterns. Overall, this model proposes a coupling between the system geometry and reaction-diffusion dynamics that can give global control over the patterning by using only local curvature information. Such a model can give shape and positioning information in animal development without the need for spatially dependent morphogen gradients.

Dynamics of BMP signaling and stable gene expression in the early Drosophila embryo.

In developing tissues, morphogen gradients are thought to initialize gene expression patterns. However, the relationship between the dynamics of morphogen-encoded signals and gene expression decisions is largely unknown. Here we examine the dynamics of the Bone Morphogenetic Protein (BMP) pathway in Drosophila blastoderm-stage embryos. In this tissue, the BMP pathway is highly dynamic: it begins as a broad and weak signal on the dorsal half of the embryo, then 20-30 min later refines into a narrow, intense peak centered on the dorsal midline. This dynamical progression of the BMP signal raises questions of how it stably activates target genes. Therefore, we performed live imaging of the BMP signal and found that dorsal-lateral cells experience only a short transient in BMP signaling, after which the signal is lost completely. Moreover, we measured the transcriptional response of the BMP target gene pannier in live embryos and found it to remain activated in dorsal-lateral cells, even after the BMP signal is lost. Our findings may suggest that the BMP pathway activates a memory, or 'ratchet' mechanism that may sustain gene expression.

Diffusion barriers imposed by tissue topology shape Hedgehog morphogen gradients

Animals use a small number of morphogens to pattern tissues, but it is unclear how evolution modulates morphogen signaling range to match tissues of varying sizes. Here, we used single-molecule imaging in reconstituted morphogen gradients and in tissue explants to determine that Hedgehog diffused extracellularly as a monomer, and rapidly transitioned between membrane-confined and -unconfined states. Unexpectedly, the vertebrate-specific protein SCUBE1 expanded Hedgehog gradients by accelerating the transition rates between states without affecting the relative abundance of molecules in each state. This observation could not be explained under existing models of morphogen diffusion. Instead, we developed a topology-limited diffusion model in which cell-cell gaps create diffusion barriers, which morphogens can only overcome by passing through a membrane-unconfined state. Under this model, SCUBE1 promoted Hedgehog secretion and diffusion by allowing it to transiently overcome diffusion barriers. This multiscale understanding of morphogen gradient formation unified prior models and identified knobs that nature can use to tune morphogen gradient sizes across tissues and organisms.

Setting the stage for embryo segmentation

Morphogen gradients are critical regulators of embryonic development. In this issue, Liu etal.1 introduce a microfluidic system that externally applies morphogen gradients to an invitro model of human embryo segmentation.It enables the investigation of signaling gradients during this developmental processat unprecedented levels of precision.

Multiple dynamic modes of Bicoid morphogen gradient are explained by a quantum-classical model

Multiple dynamic modes of Bicoid morphogen gradient are explained by a quantum-classical model

Using Microfluidics to Align Matrix Architecture and Generate Chemokine Gradients Promotes Directional Branching in a Model of Epithelial Morphogenesis.

The mechanical cue of fiber alignment plays a key role in the development of various tissues in the body. The ability to study the effect of these stimuli in vitro has been limited previously. Here, we present a microfluidic device capable of intrinsically generating aligned fibers using the microchannel geometry. The device also features tunable interstitial fluid flow and the ability to form a morphogen gradient. These aspects allow for the modeling of complex tissues and to differentiate cell response to different stimuli. To demonstrate the abilities of our device, we incorporated luminal epithelial cysts into our device and induced growth factor stimulation. We found the mechanical cue of fiber alignment to play a dominant role in cell elongation and the ability to form protrusions was dependent on cadherin-3. Together, this work serves as a springboard for future potential with these devices to answer questions in developmental biology and complex diseases such as cancers.

A human pluripotent stem cell-based somitogenesis model using microfluidics

Emerging human pluripotent stem cell (hPSC)-based embryo models are useful for studying human embryogenesis. Particularly, there are hPSC-based somitogenesis models using free-floating culture that recapitulate somite formation. Somitogenesis invivo involves intricately orchestrated biochemical and biomechanical events. However, none of the current somitogenesis models controls biochemical gradients or biomechanical signals in the culture, limiting their applicability to untangle complex biochemical-biomechanical interactions that drive somitogenesis. Herein, we develop a human somitogenesis model by confining hPSC-derived presomitic mesoderm (PSM) tissues in microfabricated trenches. Exogenous microfluidic morphogen gradients imposed on the PSM tissues cause axial patterning and trigger spontaneous rostral-to-caudal somite formation. A mechanical theory is developed to explain the size dependency between somites and the PSM. The microfluidic somitogenesis model is further exploited to reveal regulatory roles of cellular and tissue biomechanics in somite formation. This study presents a useful microengineered, hPSC-based model for understanding the biochemical and biomechanical events that guide somite formation.

Spatial Single-cell Analysis Decodes Cortical Layer and Area Specification.

The human cerebral cortex, pivotal for advanced cognitive functions, is composed of six distinct layers and dozens of functionally specialized areas1,2. The layers and areas are distinguished both molecularly, by diverse neuronal and glial cell subtypes, and structurally, through intricate spatial organization3,4. While single-cell transcriptomics studies have advanced molecular characterization of human cortical development, a critical gap exists due to the loss of spatial context during cell dissociation5,6,7,8. Here, we utilized multiplexed error-robust fluorescence in situ hybridization (MERFISH)9, augmented with deep-learning-based cell segmentation, to examine the molecular, cellular, and cytoarchitectural development of human fetal cortex with spatially resolved single-cell resolution. Our extensive spatial atlas, encompassing 16 million single cells, spans eight cortical areas across four time points in the second and third trimesters. We uncovered an early establishment of the six-layer structure, identifiable in the laminar distribution of excitatory neuronal subtypes by mid-gestation, long before the emergence of cytoarchitectural layers. Notably, while anterior-posterior gradients of neuronal subtypes were generally observed in most cortical areas, a striking exception was the sharp molecular border between primary (V1) and secondary visual cortices (V2) at gestational week 20. Here we discovered an abrupt binary shift in neuronal subtype specification at the earliest stages, challenging the notion that continuous morphogen gradients dictate mid-gestation cortical arealization6,10. Moreover, integrating single-nuclei RNA-sequencing and in situ whole transcriptomics revealed an early upregulation of synaptogenesis in V1-specific Layer 4 neurons, suggesting a role of synaptogenesis in this discrete border formation. Collectively, our findings underscore the crucial role of spatial relationships in determining the molecular specification of cortical layers and areas. This work not only provides a valuable resource for the field, but also establishes a spatially resolved single-cell analysis paradigm that paves the way for a comprehensive developmental atlas of the human brain.

Genetic mechanism regulating diversity in the placement of eyes on the head of animals

Despite the conservation of genetic machinery involved in eye development, there is a strong diversity in the placement of eyes on the head of animals. Morphogen gradients of signaling molecules are vital to patterning cues. During Drosophila eye development, Wingless (Wg), a ligand of Wnt/Wg signaling, is expressed anterolaterally to form a morphogen gradient to determine the eye- versus head-specific cell fate. The underlying mechanisms that regulate this process are yet to be fully understood. We characterized defective proventriculus (dve) (Drosophila ortholog of human SATB1), a K50 homeodomain transcription factor, as a dorsal eye gene, which regulates Wg signaling to determine eye versus head fate. Across Drosophila species, Dve is expressed in the dorsal head vertex region where it regulates wg transcription. Second, Dve suppresses eye fate by down-regulating retinal determination genes. Third, the dve-expressing dorsal head vertex region is important for Wg-mediated inhibition of retinal cell fate, as eliminating the Dve-expressing cells or preventing Wg transport from these dve-expressing cells leads to a dramatic expansion of the eye field. Together, these findings suggest that Dve regulates Wg expression in the dorsal head vertex, which is critical for determining eye versus head fate. Gain-of-function of SATB1 exhibits an eye fate suppression phenotype similar to Dve. Our data demonstrate a conserved role for Dve/SATB1 in the positioning of eyes on the head and the interocular distance by regulating Wg. This study provides evidence that dysregulation of the Wg morphogen gradient results in developmental defects such as hypertelorism in humans where disproportionate interocular distance and facial anomalies are reported.

Automatic design of gene regulatory mechanisms for spatial pattern formation

Gene regulatory mechanisms (GRMs) control the formation of spatial and temporal expression patterns that can serve as regulatory signals for the development of complex shapes. Synthetic developmental biology aims to engineer such genetic circuits for understanding and producing desired multicellular spatial patterns. However, designing synthetic GRMs for complex, multi-dimensional spatial patterns is a current challenge due to the nonlinear interactions and feedback loops in genetic circuits. Here we present a methodology to automatically design GRMs that can produce any given two-dimensional spatial pattern. The proposed approach uses two orthogonal morphogen gradients acting as positional information signals in a multicellular tissue area or culture, which constitutes a continuous field of engineered cells implementing the same designed GRM. To efficiently design both the circuit network and the interaction mechanisms—including the number of genes necessary for the formation of the target spatial pattern—we developed an automated algorithm based on high-performance evolutionary computation. The tolerance of the algorithm can be configured to design GRMs that are either simple to produce approximate patterns or complex to produce precise patterns. We demonstrate the approach by automatically designing GRMs that can produce a diverse set of synthetic spatial expression patterns by interpreting just two orthogonal morphogen gradients. The proposed framework offers a versatile approach to systematically design and discover complex genetic circuits producing spatial patterns.

Efficient Generation of Skin Organoids from Pluripotent Cells via Defined Extracellular Matrix Cues and Morphogen Gradients in a Spindle-Shaped Microfluidic Device.

Pluripotent stem cell-derived skin organoids (PSOs) emerge as a developmental skin model that is self-organized into multiple components, such as hair follicles. Despite their impressive complexity, PSOs are currently generated in the absence of 3D extracellular matrix (ECM) signals and have several major limitations, including an inverted anatomy (e.g., epidermis inside/dermis outside). In this work, a method is established to generate PSOs effectively in a chemically-defined 3D ECM environment. After examining various dermal ECM molecules, it is found that PSOs generated in collagen-type I (COLI) supplemented with laminin 511 (LAM511) exhibit increased growth compared to conventional free-floating conditions, but fail to induce complete skin differentiation due in part to necrosis. This problem is addressed by generating the PSOs in a 3D bioprinted spindle-shaped hydrogel device, which constrains organoid growth longitudinally. This culture system significantly reduces organoid necrosis and leads to a twofold increase in keratinocyte differentiation and an eightfold increase in hair follicle formation. Finally, the system is adapted as a microfluidic device to create asymmetrical gradients of differentiation factors and improves the spatial organization of dermal and epidermal cells. This study highlights the pivotal role of ECM and morphogen gradients in promoting and spatially-controlling skin differentiation in the PSO framework.