Morphogen Gradients Research Articles

Spatiotemporal control and modeling of morphogen delivery to induce gradient patterning of stem cell differentiation using fluidic channels.

The process of cell differentiation in a developing embryo is influenced by numerous factors, including various biological molecules whose presentation varies dramatically over space and time. These morphogens regulate cell fate based on concentration profiles, thus creating discrete populations of cells and ultimately generating large, complex tissues and organs. Recently, several in vitro platforms have attempted to recapitulate the complex presentation of extrinsic signals found in nature. However, it has been a challenge to design versatile platforms that can dynamically control morphogen gradients over extended periods of time. To address some of these issues, we introduce a platform using channels patterned in hydrogels to deliver multiple morphogens to cells in a 3D scaffold, thus creating a spectrum of cell phenotypes based on the resultant morphogen gradients. The diffusion coefficient of a common small molecule morphogen, retinoic acid (RA), was measured within our hydrogel platform using Raman spectroscopy and its diffusion in our platform's geometry was modeled using finite element analysis. The predictive model of spatial gradients was validated in a cell-free hydrogel, and temporal control of morphogen gradients was then demonstrated using a reporter cell line that expresses green fluorescent protein in the presence of RA. Finally, the utility of this approach for regulating cell phenotype was demonstrated by generating opposing morphogen gradients to create a spectrum of mesenchymal stem cell differentiation states.

Dynamic Hh signalling can generate temporal information during tissue patterning.

The differentiation of tissues and organs requires that cells exchange information in space and time. Spatial information is often conveyed by morphogens: molecules that disperse across receiving cells to generate signalling gradients. Cells translate such concentration gradients into space-dependent patterns of gene expression and cellular behaviour. But could morphogen gradients also convey developmental time? Here, by investigating the developmental role of Hh on a component of the Drosophila visual system, the ocellar retina, we have discovered that ocellar cells use the non-linear gradient of Hh as a temporal cue, collectively performing the biological equivalent of a mathematical logarithmic transformation. In this way, a morphogen diffusing from a non-moving source is decoded as a wave of differentiating photoreceptors that travels at constant speed throughout the retinal epithelium.

Molecular control of macroscopic forces drives formation of the vertebrate hindgut.

The embryonic gut tube is a cylindrical structure from which the respiratory and gastrointestinal tracts develop1. Although the early emergence of the endoderm as an epithelial sheet2,3 and later morphogenesis of the definitive digestive and respiratory organs4-6 have been investigated, the intervening process of gut tube formation remains relatively understudied7,8. Here we investigate the molecular control of macroscopic forces underlying early morphogenesis of the gut tube in the chick embryo. The gut tube has been described as forming from two endodermal invaginations-the anterior intestinal portal (AIP) towards the rostral end of the embryo and the caudal intestinal portal (CIP) at the caudal end-that migrate towards one another, internalizing the endoderm until they meet at the yolk stalk (umbilicus in mammals)1,6. Migration of the AIP to form foregut has been descriptively characterized8,9, but the hindgut is likely to form by a distinct mechanism that has not been fully explained10. We find that the hindgut is formed by collective cell movements through a stationary CIP, rather than by movement of the CIP itself. Further, combining in vivo imaging, biophysics and mathematical modelling with molecular and embryological approaches, we identify a contractile force gradient that drives cell movements in the hindgut-forming endoderm, enabling tissue-scale posterior extension of the forming hindgut tube. The force gradient, in turn, is established in response to a morphogenic gradient of fibroblast growth factor signalling. As a result, we propose that an important positive feedback arises, whereby contracting cells draw passive cells from low to high fibroblast growth factor levels, recruiting them to contract and pull more cells into the elongating hindgut. In addition to providing insight into the early gut development, these findings illustrate how large-scale tissue level forces can be traced to developmental signals during vertebrate morphogenesis.

Some chemotactic mutants can be progress through development in chimeric populations

Cell migration in response to morphogen gradients affects morphogenesis. Chemotaxis towards adenosine 3', 5'-monophosphate (cAMP) is essential for the early stage of morphogenesis in the slime mold Dictyostelium discoideum. Here, we show that D. discoideum completes morphogenesis without cAMP-chemotaxis-dependent cell migration. The extracellular cAMP gradient is believed to cause cells to form a slug-shaped multicellular structure and fruiting body. The cAMP receptor, cAR1, was not expressed at the cell surface during these stages, correlating with reduced chemotactic activity. Gβ-null cells expressing temperature sensitive Gβ are unable to generate extracellular cAMP (Jin et al., 1998) and thus unable to aggregate and exhibit proper morphogenesis under restrictive temperature. However, when mixed with wild type cells ts-Gβ expressing gβ-null cells normally aggregated and exhibited normal morphogenesis under restrictive temperature. Furthermore, cells migrated after aggregation in a mixture containing wild-type cells. KI-5 cells, which do not show aggregation or morphogenesis, spontaneously migrated to a transplanted wild-type tip and underwent normal morphogenesis and cell differentiation; this was not observed in cells lacking tgrB1and tgrC1 cells adhesion molecules. Thus, cAMP gradient-dependent cell migration may not be required for multicellular pattern formation in late Dictyostelium development.

Entrainment as a means of controlling phase waves in populations of coupled oscillators

We explore waves and entrainment in a model of coupled oscillators, inspired from the cellular oscillators in the presomitic mesoderm (PSM) of mice. The internal clock in each cell is based on a negative feedback loop which couples to the clocks of neighboring cells through a Notch mechanism. We investigate how a morphogen gradient in the mesoderm, which affects the period of oscillating cells, gives rise to phase waves traveling from the posterior to the anterior part of the PSM. We show that the phase waves can be entrained by an external periodic variation in this morphogen and also observe that multiple oscillatory solutions can coexist in the cell population. Together, these provide a way to potentially control phase waves and thereby manipulate somite patterning in embryos, based on entrainment properties of coupled nonlinear oscillators.

A re-inducible gap gene cascade patterns the anterior-posterior axis of insects in a threshold-free fashion.

Gap genes mediate the division of the anterior-posterior axis of insects into different fates through regulating downstream hox genes. Decades of tinkering the segmentation gene network of Drosophila melanogaster led to the conclusion that gap genes are regulated (at least initially) through a threshold-based mechanism, guided by both anteriorly- and posteriorly-localized morphogen gradients. In this paper, we show that the response of the gap gene network in the beetle Tribolium castaneum upon perturbation is consistent with a threshold-free 'Speed Regulation' mechanism, in which the speed of a genetic cascade of gap genes is regulated by a posterior morphogen gradient. We show this by re-inducing the leading gap gene (namely, hunchback) resulting in the re-induction of the gap gene cascade at arbitrary points in time. This demonstrates that the gap gene network is self-regulatory and is primarily under the control of a posterior regulator in Tribolium and possibly other short/intermediate-germ insects.

Around the clock: gradient shape and noise impact the evolution of oscillatory segmentation dynamics

BackgroundSegmentation, the subdivision of the major body axis into repeated elements, is considered one of the major evolutionary innovations in bilaterian animals. In all three segmented animal clades, the predominant segmentation mechanism is sequential segmentation, where segments are generated one by one in anterior–posterior order from a posterior undifferentiated zone. In vertebrates and arthropods, sequential segmentation is thought to arise from a clock-and-wavefront-type mechanism, where oscillations in the posterior growth zone are transformed into a segmental prepattern in the anterior by a receding wavefront. Previous evo-devo simulation studies have demonstrated that this segmentation type repeatedly arises, supporting the idea of parallel evolutionary origins in these animal clades. Sequential segmentation has been studied most extensively in vertebrates, where travelling waves have been observed that reflect the slowing down of oscillations prior to their cessation and where these oscillations involve a highly complex regulatory network. It is currently unclear under which conditions this oscillator complexity and slowing should be expected to evolve, how they are related and to what extent similar properties should be expected for sequential segmentation in other animal species.ResultsTo investigate these questions, we extend a previously developed computational model for the evolution of segmentation. We vary the slope of the posterior morphogen gradient and the strength of gene expression noise. We find that compared to a shallow gradient, a steep morphogen gradient allows for faster evolution and evolved oscillator networks are simpler. Furthermore, under steep gradients, damped oscillators often evolve, whereas shallow gradients appear to require persistent oscillators which are regularly accompanied by travelling waves, indicative of a frequency gradient. We show that gene expression noise increases the likelihood of evolving persistent oscillators under steep gradients and of evolving frequency gradients under shallow gradients. Surprisingly, we find that the evolutions of oscillator complexity and travelling waves are not correlated, suggesting that these properties may have evolved separately.ConclusionsBased on our findings, we suggest that travelling waves may have evolved in response to shallow morphogen gradients and gene expression noise. These two factors may thus also be responsible for the observed differences between different species within both the arthropod and chordate phyla.

Signalling and differentiation in emulsion-based multi-compartmentalized in vitro gene circuits

Multicellularity enables the growth of complex life forms as it allows for specialization of cell types, differentiation, and large scale spatial organization. In a similar way, modular construction of synthetic multicellular systems will lead to dynamic biomimetic materials that can respond to their environment in complex ways. In order to achieve this goal, artificial cellular communication and developmental programs still have to be established. Here, we create geometrically controlled spatial arrangements of emulsion-based artificial cellular compartments containing synthetic in vitro gene circuitry, separated by lipid bilayer membranes. We quantitatively determine the membrane pore-dependent response of the circuits to artificial morphogen gradients, which are established via diffusion from dedicated organizer cells. Utilizing different types of feed-forward and feedback in vitro gene circuits, we then implement artificial signaling and differentiation processes, demonstrating the potential for the realization of complex spatiotemporal dynamics in artificial multicellular systems.

Imaging and Quantification of P-Smad1/5 in Zebrafish Blastula and Gastrula Embryos.

Spatiotemporal patterns of morphogen activity drive differential gene expression with a high degree of precision within a developing embryo and reproducibly between embryos. Understanding the formation and function of a morphogen gradient during development requires quantitative measurement of morphogen activity throughout an individual embryo and also between embryos within a population. Quantification of morphogen gradients in to presents unique challenges in imaging and image processing to minimize error and maximize the quality of the data so it may be used in computational models of development and in statistically testing hypotheses. Here we present methods for the preparation, immunostaining, imaging, and quantification of a bone morphogenetic protein (BMP) activity gradient in individual zebrafish embryos as well as methods for acquiring population-level statistics after embryo grouping and alignment. This quantitative approach can be extended to other morphogen systems, and the computational codes can be adapted to other imaging contexts in zebrafish and other organisms.

3 minutes to precisely measure morphogen concentration.

Morphogen gradients provide concentration-dependent positional information along polarity axes. Although the dynamics of the establishment of these gradients is well described, precision and noise in the downstream activation processes remain elusive. A simple paradigm to address these questions is the Bicoid morphogen gradient that elicits a rapid step-like transcriptional response in young fruit fly embryos. Focusing on the expression of the major Bicoid target, hunchback (hb), at the onset of zygotic transcription, we used the MS2-MCP approach which combines fluorescent labeling of nascent mRNA with live imaging at high spatial and temporal resolution. Removing 36 putative Zelda binding sites unexpectedly present in the original MS2 reporter, we show that the 750 bp of the hb promoter are sufficient to recapitulate endogenous expression at the onset of zygotic transcription. After each mitosis, in the anterior, expression is turned on to rapidly reach a plateau with all nuclei expressing the reporter. Consistent with a Bicoid dose-dependent activation process, the time period required to reach the plateau increases with the distance to the anterior pole. Despite the challenge imposed by frequent mitoses and high nuclei-to-nuclei variability in transcription kinetics, it only takes 3 minutes at each interphase for the MS2 reporter loci to distinguish subtle differences in Bicoid concentration and establish a steadily positioned and steep (Hill coefficient ~ 7) expression boundary. Modeling based on the cooperativity between the 6 known Bicoid binding sites in the hb promoter region, assuming rate limiting concentrations of the Bicoid transcription factor at the boundary, is able to capture the observed dynamics of pattern establishment but not the steepness of the boundary. This suggests that a simple model based only on the cooperative binding of Bicoid is not sufficient to describe the spatiotemporal dynamics of early hb expression.

Local Auxin Biosynthesis Is a Key Regulator of Plant Development

Auxin is a major phytohormone that controls numerous aspects of plant development and coordinates plant responses to the environment. Morphogenic gradients of auxin govern cell fate decisions and underlie plant phenotypic plasticity. Polar auxin transport plays a central role in auxin maxima generation. The discovery of the exquisite spatiotemporal expression patterns of auxin biosynthesis genes of the WEI8/TAR and YUC families suggested that local auxin production may contribute to the formation of auxin maxima. Herein, we systematically addressed the role of local auxin biosynthesis in plant development and responses to the stress phytohormone ethylene by manipulating spatiotemporal patterns of WEI8. Our study revealed that local auxin biosynthesis and transport act synergistically and are individually dispensable for root meristem maintenance. In contrast, flower fertility and root responses to ethylene require local auxin production that cannot be fully compensated for by transport in the generation of morphogenic auxin maxima.

Feedback regulation of cytoneme-mediated transport shapes a tissue-specific FGF morphogen gradient.

Gradients of signaling proteins are essential for inducing tissue morphogenesis. However, mechanisms of gradient formation remain controversial. Here we characterized the distribution of fluorescently-tagged signaling proteins, FGF and FGFR, expressed at physiological levels from the genomic knock-in alleles in Drosophila. FGF produced in the larval wing imaginal-disc moves to the air-sac-primordium (ASP) through FGFR-containing cytonemes that extend from the ASP to contact the wing-disc source. The number of FGF-receiving cytonemes extended by ASP cells decreases gradually with increasing distance from the source, generating a recipient-specific FGF gradient. Acting as a morphogen in the ASP, FGF activates concentration-dependent gene expression, inducing pointed-P1 at higher and cut at lower levels. The transcription-factors Pointed-P1 and Cut antagonize each other and differentially regulate formation of FGFR-containing cytonemes, creating regions with higher-to-lower numbers of FGF-receiving cytonemes. These results reveal a robust mechanism where morphogens self-generate precise tissue-specific gradient contours through feedback regulation of cytoneme-mediated dispersion.

SNX3-retromer requires an evolutionary conserved MON2:DOPEY2:ATP9A complex to mediate Wntless sorting and Wnt secretion

Wntless transports Wnt morphogens to the cell surface and is required for Wnt secretion and morphogenic gradients formation. Recycling of endocytosed Wntless requires the sorting nexin-3 (SNX3)-retromer-dependent endosome-to-Golgi transport pathway. Here we demonstrate the essential role of SNX3-retromer assembly for Wntless transport and report that SNX3 associates with an evolutionary conserved endosome-associated membrane re-modelling complex composed of MON2, DOPEY2 and the putative aminophospholipid translocase, ATP9A. In vivo suppression of Ce-mon-2, Ce-pad-1 or Ce-tat-5 (respective MON2, DOPEY2 and ATP9A orthologues) phenocopy a loss of SNX3-retromer function, leading to enhanced lysosomal degradation of Wntless and a Wnt phenotype. Perturbed Wnt signalling is also observed upon overexpression of an ATPase-inhibited TAT-5(E246Q) mutant, suggesting a role for phospholipid flippase activity during SNX3-retromer-mediated Wntless sorting. Together, these findings provide in vitro and in vivo mechanistic details to describe SNX3-retromer-mediated transport during Wnt secretion and the formation of Wnt-morphogenic gradients.

Knockdown of Mns1 Increases Susceptibility to Craniofacial Defects Following Gastrulation-Stage Alcohol Exposure in Mice.

MNS1 (meiosis-specific nuclear structural protein 1) is necessary for motile cilia function, such as sperm flagella or those found in the embryonic primitive node. While little is known regarding the function or expression pattern of MNS1 in the embryo, co-immunoprecipitation experiments in sperm have determined that MNS1 interacts with ciliary proteins, which are also important during development. Establishment of morphogenic gradients is dependent on normal ciliary motion in the primitive node beginning during gastrulation (gestational day [GD] 7 in the mouse, second-third week of pregnancy in humans), a critical window for face, eye, and brain development and particularly susceptible to perturbations of developmental signals. The current study investigates the role of Mns1 in craniofacial defects associated with gastrulation-stage alcohol exposure. On GD7, pregnant Mns1+/- dams were administered 2 doses of ethanol (5.8g/kg total) or vehicle 4hours apart to target gastrulation. On GD17, fetuses were examined for ocular defects by scoring each eye on a scale from 1 to 7 (1=normal, 2 to 7=defects escalating in severity). Craniofacial and brain abnormalities were also assessed. Prenatal alcohol exposure (PAE) significantly increased the rate of defects in wild-type fetuses, as PAE fetuses had an incidence rate of 41.18% compared to a 10% incidence rate in controls. Furthermore, PAE interacted with genotype to significantly increase the defect rate and severity in Mns1+/- (64.29%) and Mns1-/- mice (92.31%). PAE Mns1-/- fetuses with severe eye defects also presented with craniofacial dysmorphologies characteristic of fetal alcohol syndrome and midline tissue loss in the brain, palate, and nasal septum. These data demonstrate that a partial or complete knockdown of Mns1 interacts with PAE to increase the susceptibility to ocular defects and correlating craniofacial and brain anomalies, likely though interaction of alcohol with motile cilia function. These results further our understanding of genetic risk factors that may underlie susceptibility to teratogenic exposures.

The Role of the Primary Cell Wall in Plant Morphogenesis.

Morphogenesis remains a riddle, wrapped in a mystery, inside an enigma. It remains a formidable problem viewed from many different perspectives of morphology, genetics, and computational modelling. We propose a biochemical reductionist approach that shows how both internal and external physical forces contribute to plant morphogenesis via mechanical stress–strain transduction from the primary cell wall tethered to the plasma membrane by a specific arabinogalactan protein (AGP). The resulting stress vector, with direction defined by Hechtian adhesion sites, has a magnitude of a few piconewtons amplified by a hypothetical Hechtian growth oscillator. This paradigm shift involves stress-activated plasma membrane Ca2+ channels and auxin-activated H+-ATPase. The proton pump dissociates periplasmic AGP-glycomodules that bind Ca2+. Thus, as the immediate source of cytosolic Ca2+, an AGP-Ca2+ capacitor directs the vectorial exocytosis of cell wall precursors and auxin efflux (PIN) proteins. In toto, these components comprise the Hechtian oscillator and also the gravisensor. Thus, interdependent auxin and Ca2+ morphogen gradients account for the predominance of AGPs. The size and location of a cell surface AGP-Ca2+ capacitor is essential to differentiation and explains AGP correlation with all stages of morphogenetic patterning from embryogenesis to root and shoot. Finally, the evolutionary origins of the Hechtian oscillator in the unicellular Chlorophycean algae reflect the ubiquitous role of chemiosmotic proton pumps that preceded DNA at the dawn of life.

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.

An essential role for the nuclear protein Akirin2 in mouse limb interdigital tissue regression

The regulation of interdigital tissue regression requires the interplay of multiple spatiotemporally-controlled morphogen gradients to ensure proper limb formation and release of individual digits. Disruption to this process can lead to a number of limb abnormalities, including syndactyly. Akirins are highly conserved nuclear proteins that are known to interact with chromatin remodelling machinery at gene enhancers. In mammals, the analogue Akirin2 is essential for embryonic development and critical for a wide variety of roles in immune function, meiosis, myogenesis and brain development. Here we report a critical role for Akirin2 in the regulation of interdigital tissue regression in the mouse limb. Knockout of Akirin2 in limb epithelium leads to a loss of interdigital cell death and an increase in cell proliferation, resulting in retention of the interdigital web and soft-tissue syndactyly. This is associated with perdurance of Fgf8 expression in the ectoderm overlying the interdigital space. Our study supports a mechanism whereby Akirin2 is required for the downregulation of Fgf8 from the apical ectodermal ridge (AER) during limb development, and implies its requirement in signalling between interdigital mesenchymal cells and the AER.

A Local Auxin Gradient Regulates Root Cap Self-Renewal and Size Homeostasis

Organ size homeostasis, compensatory growth to replace lost tissue, requires constant measurement of size and adjustment of growth rates. Morphogen gradients control organ and tissue sizes by regulating stem cell activity, cell differentiation, and removal in animals [1-3]. In plants, control of tissue size is of specific importance in root caps to protect the growing root tip from mechanical damage [4]. New root cap tissue is formed by the columella and lateral root-cap-epidermal stem cells, whose activity is regulated through non-dividing niche-like cells, the quiescent center (QC) [4, 5]. Columella daughter cells in contact with the QC retain the potency to divide, while derivatives oriented toward the mature cap undergo differentiation. The outermost columella layers are sequentially separated from the root body, involving remodeling of cell walls [6]. Factors regulating the balance between cell division, elongation, and separation to keep root cap size constant are currently unknown [4]. Here, we report that stem cell proliferation induced cell separation at the periphery of the root cap, resulting in tissue size homeostasis. An auxin response gradient with a maximum in the QC and a minimum in the detaching layer was established prior to the onset of cell separation. In agreement with a mathematical model, tissue size was positively regulated by the amount of auxin released from the source. Auxin transporters localized non-polarly to plasma membranes of the inner cap, partly isolating separating layers from the auxin source. Together, these results are in support of an auxin gradient measuring and regulating tissue size.

In situ-forming, cell-instructive hydrogels based on glycosaminoglycans with varied sulfation patterns

Glycosaminoglycan (GAG)-based hydrogels were proven highly effective to direct cell fate decisions by modulating the administration of cytokines. The sulfation pattern of the GAG component critically controls its affinity to proteins and thus governs the release of cytokines from GAG-containing gel systems. To apply this principle in the design of in situ assembling materials suitable for cell embedding and injection into tissues, we developed a platform of bio-orthogonally crosslinked star-shaped poly(ethylene glycol) (starPEG)-GAG hydrogels that display variable GAG sulfation patterns. Combining rational design for tuning the hydrogel network properties and a reaction-diffusion model for predicting transport processes within the matrices, we exemplarily applied the resulting materials for tailoring morphogenic and chemotactic gradients of platelet-derived growth factor-BB (PDGF-BB) in 3D. Conditions identified with this approach were demonstrated to effectively control the fate and morphogenesis of embedded mesenchymal stem cells (MSCs). Adjusting the sulfation patterns of glycosamnioglycans used in the preparation of in situ forming hydrogels is thus concluded to create new powerful options for modulating biomolecular signals in cell fate control, paving the way for advanced 3D cultures and precision tissue engineering.

Developmental Biology: Morphogen in a Dish

Reconstitution of a Hedgehog morphogen gradient invitro and in silico reveals the architectural features of the signal transduction pathway that ensure rapid formation of a robust signalling gradient.