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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. Synopsis: A synthetic biology approach termed SYMPLE3D demonstrates that cell adhesion output in response to diffusible proteins can convert a signaling gradient into binarized tissue domains with a sharp boundary. The SYMPLE3D system allows to explore which cellular responses to secreted proteins can robustly generate multicellular patterns. Interlocking of morphogen gradient and cadherin induces a robust pattern of distinct tissue domains with a sharp boundary. Morphogen-induced cadherin gathers receiver cells to remove ectopically-activated cells within a signaling gradient. A switch-like property of cadherin function causes cell mixing inside a morphogen gradient, generating a uniformly-activated tissue domain. A synthetic biology approach termed SYMPLE3D demonstrates that cell adhesion output in response to diffusible proteins can convert a signaling gradient into binarized tissue domains with a sharp boundary. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Receptor tyrosine kinases exhibit ligand‐induced activity and uptake into cells via endocytosis. In the case of epidermal growth factor (EGF) receptor (EGFR), the resulting endosomes are trafficked to the perinuclear region, where dephosphorylation of receptors occurs, which are subsequently directed to degradation. Traveling endosomes bearing phosphorylated EGFRs are subjected to the activity of cytoplasmic phosphatases as well as interactions with the endoplasmic reticulum (ER). The peri‐nuclear region harbors ER‐embedded phosphatases, a component of the EGFR‐bearing endosome‐ER contact site. The ER is also emerging as a central player in spatiotemporal control of endosomal motility, positioning, tubulation, and fission. Past studies strongly suggest that the physical interaction between the ER and endosomes forms a reaction "unit" for EGFR dephosphorylation. Independently, endosomes have been implicated to enable quantization of EGFR signals by modulation of the phosphorylation levels. Here, we review the distinct mechanisms by which endosomes form the logistical means for signal quantization and speculate on the role of the ER. [ABSTRACT FROM AUTHOR]

Thermal plasticity of melanin pigmentation patterns in Drosophila species has been studied as a model to investigate developmental mechanisms of phenotypic plasticity. The developmental process of melanin pigmentation patterns on wings of Drosophila is divided into two parts, prepattern specification during the pupal period and wing vein-dependent transportation of melanin precursors after eclosion. Which part can be affected by thermal changes? To address this question, we used polka-dotted melanin spots on wings of Drosophila guttifera, whose spot areas are specified by wingless morphogen. In this research, we reared D. guttifera at different temperatures to test whether wing spots show thermal plasticity. We found that wing size becomes larger at lower temperature and that different spots have different reaction norms. Furthermore, we changed the rearing temperature in the middle of the pupal period and found that the most sensitive developmental periods for wing size and spot size are different. The results suggest that the size control mechanisms for the thermal plasticity of wing size and spot size are independent. We also found that the most sensitive stage for spot size was part of the pupal period including stages at which wingless is expressed in the polka-dotted pattern. Therefore, it is suggested that temperature change might affect the prepattern specification process and might not affect transportation through wing veins.

Cortical arealization arises during neurodevelopment from the confluence of molecular gradients representing patterned expression of morphogens and transcription factors. However, whether similar gradients are maintained in the adult brain remains unknown. Here, we uncover three axes of topographic variation in gene expression in the adult human brain that specifically capture previously identified rostral-caudal, dorsal-ventral, and medial-lateral axes of early developmental patterning. The interaction of these spatiomolecular gradients i) accurately reconstructs the position of brain tissue samples, ii) delineates known functional territories, and iii) can model the topographical variation of diverse cortical features. The spatiomolecular gradients are distinct from canonical cortical axes differentiating the primary sensory cortex from the association cortex, but radiate in parallel with the axes traversed by local field potentials along the cortex. We replicate all three molecular gradients in three independent human datasets as well as two nonhuman primate datasets and find that each gradient shows a distinct developmental trajectory across the lifespan. The gradients are composed of several well-known transcription factors (e.g., PAX6 and SIX3), and a small set of genes shared across gradients are strongly enriched for multiple diseases. Together, these results provide insight into the developmental sculpting of functionally distinct brain regions, governed by three robust transcriptomic axes embedded within brain parenchyma. [ABSTRACT FROM AUTHOR]

Wnt, a family of secreted signaling proteins, serves diverse functions in embryogenesis, organogenesis, cancer, and stem cell functions. In the context of development, Wnt has been considered a representative morphogen, forming concentration gradients to give positional information to cells or tissues. However, although gradients are often illustrated in schemata, the reality of concentration gradients, or in other words, actual spatial distribution of Wnt ligands, and their behaviors in the extracellular space still remain poorly known. To understand extracellular behavior of Wnt ligands, quantitative analyses such as fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) are highly informative because Wnt dispersal involves physical and biochemical processes, such as diffusion and binding to or dissociation from cell surface molecules, including heparan sulfate proteoglycans (HSPGs). Here, I briefly discuss representative methods to quantify morphogen dynamics. In addition, I discuss molecular manipulations of morphogens, mainly focusing on use of protein binders, and synthetic biology of morphogens as indicators of current and future directions in this field. [ABSTRACT FROM AUTHOR]

Wnt is a family of secreted signaling proteins involved in the regulation of cellular processes, including maintenance of stem cells, carcinogenesis, and cell differentiation. In the context of early vertebrate embryogenesis, graded distribution of Wnt proteins has been thought to regulate positional information along the antero‐posterior axis. However, understanding of the molecular basis for Wnt spatial distribution remains poor. Modified states of heparan sulfate (HS) proteoglycans are essential for Wnt8 localization, because depletion of N‐deacetylase/N‐sulfotransferase 1 (NDST1), a modification enzyme of HS chains, decreases Wnt8 levels and NDST1 overexpression increases Wnt8 levels on the cell surface. Since overexpression of NDST1 increases both deacetylation and N‐sulfation of HS chains, it is not clear which function of NDST1 is actually involved in Wnt8 localization. In the present study, we generated an NDST1 mutant that specifically increases deacetylation, but not N‐sulfation, of HS chains in Xenopus embryos. Unlike wild‐type NDST1, this mutant did not increase Wnt8 accumulation on the cell surface, but it reduced canonical Wnt signaling, as determined with the TOP‐Flash reporter assay. These results suggest that N‐sulfation of HS chains is responsible for localization of Wnt8 and Wnt8 signaling, whereas deacetylation has an inhibitory effect on canonical Wnt signaling. Consistently, overexpression of wild‐type NDST1, but not the mutant, resulted in small eyes in Xenopus embryos. Thus, our NDST1 mutant enables us to dissect the regulation of Wnt8 localization and signaling by HS proteoglycans by specifically manipulating the enzymatic activities of NDST1. [ABSTRACT FROM AUTHOR]

Introduction: Plant growth is a plastic phenomenon controlled both by endogenous genetic programs and by environmental cues. The embryonic stem, the hypocotyl, is an ideal model system for the quantitative study of growth due to its relatively simple geometry and cellular organization, and to its essentially unidirectional growth pattern. The hypocotyl of Arabidopsis thaliana has been studied particularly well at the molecular-genetic level and at the cellular level, and it is the model of choice for analysis of the shade avoidance syndrome (SAS), a growth reaction that allows plants to compete with neighboring plants for light. During SAS, hypocotyl growth is controlled primarily by the growth hormone auxin, which stimulates cell expansion without the involvement of cell division. Methods: We assessed hypocotyl growth at cellular resolution in Arabidopsis mutants defective in auxin transport and biosynthesis and we designed a mathematical auxin transport model based on known polar and non-polar auxin transporters (ABCB1, ABCB19, and PINs) and on factors that control auxin homeostasis in the hypocotyl. In addition, we introduced into the model biophysical properties of the cell types based on precise cell wall measurements. Results and Discussion: Our model can generate the observed cellular growth patterns based on auxin distribution along the hypocotyl resulting from production in the cotyledons, transport along the hypocotyl, and general turnover of auxin. These principles, which resemble the features of mathematical models of animal morphogen gradients, allow to generate robust shallow auxin gradients as they are expected to exist in tissues that exhibit quantitative auxin-driven tissue growth, as opposed to the sharp auxin maxima generated by patterning mechanisms in plant development. [ABSTRACT FROM AUTHOR]

Dpp/BMP acts as a morphogen to provide positional information in the Drosophila wing disc. Key cell-surface molecules to control Dpp morphogen gradient formation and signaling are heparan sulfate proteoglycans (HSPGs). In the wing disc, two HSPGs, the glypicans Division abnormally delayed (Dally) and Dally-like (Dlp) have been suggested to act redundantly to control these processes through direct interaction of their heparan sulfate (HS) chains with Dpp. Based on this assumption, a number of models on how glypicans control Dpp gradient formation and signaling have been proposed, including facilitating or hindering Dpp spreading, stabilizing Dpp on the cell surface, or recycling Dpp. However, how distinct HSPGs act remains largely unknown. Here, we generate genome-engineering platforms for the two glypicans and find that only Dally is critical for Dpp gradient formation and signaling through interaction of its core protein with Dpp. We also find that this interaction is not sufficient and that the HS chains of Dally are essential for these functions largely without interacting with Dpp. We provide evidence that the HS chains of Dally are not essential for spreading or recycling of Dpp but for stabilizing Dpp on the cell surface by antagonizing receptor-mediated Dpp internalization. These results provide new insights into how distinct HSPGs control morphogen gradient formation and signaling during development.

Morphogen gradients impart positional information to cells in a homogenous tissue field. Fgf8a, a highly conserved growth factor, has been proposed to act as a morphogen during zebrafish gastrulation. However, technical limitations have so far prevented direct visualization of the endogenous Fgf8a gradient and confirmation of its morphogenic activity. Here, we monitor Fgf8a propagation in the developing neural plate using a CRISPR/Cas9-mediated EGFP knock-in at the endogenous fgf8a locus. By combining sensitive imaging with single-molecule fluorescence correlation spectroscopy, we demonstrate that Fgf8a, which is produced at the embryonic margin, propagates by diffusion through the extracellular space and forms a graded distribution towards the animal pole. Overlaying the Fgf8a gradient curve with expression profiles of its downstream targets determines the precise input-output relationship of Fgf8a-mediated patterning. Manipulation of the extracellular Fgf8a levels alters the signaling outcome, thus establishing Fgf8a as a bona fide morphogen during zebrafish gastrulation. Furthermore, by hindering Fgf8a diffusion, we demonstrate that extracellular diffusion of the protein from the source is crucial for it to achieve its morphogenic potential. [ABSTRACT FROM AUTHOR]

Intercellular signaling molecules, known as morphogens, act at a long range in developing tissues to provide spatial information and control properties such as cell fate and tissue growth. The production, transport, and removal of morphogens shape their concentration profiles in time and space. Downstream signaling cascades and gene regulatory networks within cells then convert the spatiotemporal morphogen profiles into distinct cellular responses. Current challenges are to understand the diverse molecular and cellular mechanisms underlying morphogen gradient formation, as well as the logic of downstream regulatory circuits involved in morphogen interpretation. This knowledge, combining experimental and theoretical results, is essential to understand emerging properties of morphogen-controlled systems, such as robustness and scaling. [ABSTRACT FROM AUTHOR]

Secreted signaling molecules act as morphogens to control patterning and growth in many developing tissues. Since locally produced morphogens spread to form a concentration gradient in the surrounding tissue, spreading is generally thought to be the key step in the non‐autonomous actions. Here, we review recent advances in tool development to investigate morphogen function using the role of decapentaplegic (Dpp)/bone morphogenetic protein (BMP)‐type ligand in the Drosophila wing disc as an example. By applying protein binder tools to distinguish between the roles of Dpp spreading and local Dpp signaling, we found that Dpp signaling in the source cells is important for wing patterning and growth but Dpp spreading from this source cells is not as strictly required as previously thought. Given recent studies showing unexpected requirements of long‐range action of different morphogens, manipulating endogenous morphogen gradients by synthetic protein binder tools could shed more light on how morphogens act in developing tissues. [ABSTRACT FROM AUTHOR]

A ubiquitous feature of the auditory organ in amniotes is the longitudinal mapping of neuronal characteristic frequencies (CFs), which increase exponentially with distance along the organ. The exponential tonotopic map reflects variation in hair cell properties according to cochlear location and is thought to stem from concentration gradients in diffusible morphogenic proteins during embryonic development. While in all amniotes the spatial gradient is initiated by sonic hedgehog (SHH), released from the notochord and floorplate, subsequent molecular pathways are not fully understood. In chickens, BMP7 is one such morphogen, secreted from the distal end of the cochlea. In mammals, the developmental mechanism differs from birds and may depend on cochlear location. A consequence of exponential maps is that each octave occupies an equal distance on the cochlea, a spacing preserved in the tonotopic maps in higher auditory brain regions. This may facilitate frequency analysis and recognition of acoustic sequences. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Hs6st1 is expressed in lateral sensorial layer of neuroectoderm in Xenopus neurula. • Hs6st1 organizes neuroectodermal patterning and subsequent retinal formation. • 6-O-sulfation of heparan sulfate mediated by Hs6st1 accumulates Fgf8a but not Noggin. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In vertebrates with elongated auditory organs, mechanosensory hair cells (HCs) are organised such that complex sounds are broken down into their component frequencies along a proximal-to-distal long (tonotopic) axis. Acquisition of unique morphologies at the appropriate position along the chick cochlea, the basilar papilla, requires that nascent HCs determine their tonotopic positions during development. The complex signalling within the auditory organ between a developing HC and its local niche along the cochlea is poorly understood. Using a combination of live imaging and NAD(P)H fluorescence lifetime imaging microscopy, we reveal that there is a gradient in the cellular balance between glycolysis and the pentose phosphate pathway in developing HCs along the tonotopic axis. Perturbing this balance by inhibiting different branches of cytosolic glucose catabolism disrupts developmental morphogen signalling and abolishes the normal tonotopic gradient in HC morphology. These findings highlight a causal link between graded morphogen signalling and metabolic reprogramming in specifying the tonotopic identity of developing HCs.

During epithelium tissue maintenance, lineages of cells differentiate and proliferate in a coordinated way to provide the desirable size and spatial organization of different types of cells. While mathematical models through deterministic description have been used to dissect role of feedback regulations on tissue layer size and stratification, how the stochastic effects influence tissue maintenance remains largely unknown. Here we present a stochastic continuum model for cell lineages to investigate how both layer thickness and layer stratification are affected by noise. We find that the cell-intrinsic noise often causes reduction and oscillation of layer size whereas the cell-extrinsic noise increases the thickness, and sometimes, leads to uncontrollable growth of the tissue layer. The layer stratification usually deteriorates as the noise level increases in the cell lineage systems. Interestingly, the morphogen noise, which mixes both cell-intrinsic noise and cell-extrinsic noise, can lead to larger size of layer with little impact on the layer stratification. By investigating different combinations of the three types of noise, we find the layer thickness variability is reduced when cell-extrinsic noise level is high or morphogen noise level is low. Interestingly, there exists a tradeoff between low thickness variability and strong layer stratification due to competition among the three types of noise, suggesting robust layer homeostasis requires balanced levels of different types of noise in the cell lineage systems.

BackgroundSpatial pattern formation is a critical issue in developmental biology. Gene expression boundary sharpening has been observed from both experiments and modeling simulations. However, the mechanism to determine the sharpness of the boundary is not fully elucidated.ResultsWe investigated the boundary sharpening resulted by three biological motifs, interacting with morphogens, and uncovered their probabilistic landscapes. The landscape view, along with calculated average switching time between attractors, provides a natural explanation for the boundary sharpening behavior relying on the noise induced gene state switchings. To possess boundary sharpening potential, a gene network needs to generate an asymmetric bistable state, i.e. one of the two stable states is less stable than the other. We found that the mutual repressed self-activation model displays more robust boundary sharpening ability against parameter perturbation, compared to the mutual repression or the self-activation model. This is supported by the results of switching time calculated from the landscape, which indicate that the mutual repressed self-activation model has shortest switching time, among three models. Additionally, introducing cross gradients of morphogens provides a more stable mechanism for the boundary sharpening of gene expression, due to a two-way switching mechanism.ConclusionsOur results reveal the underlying principle for the gene expression boundary sharpening, and pave the way for the mechanistic understanding of cell fate decisions in the pattern formation processes of development.

Pattern formation is the process by which cells within a homogeneous epithelial sheet acquire distinctive fates depending upon their relative spatial position to each other. Several proposals, starting with Alan Turing’s diffusion-reaction model, have been put forth over the last 70 years to describe how periodic patterns like those of vertebrate somites and skin hairs, mammalian molars, fish scales, and avian feather buds emerge during development. One of the best experimental systems for testing said models and identifying the gene regulatory networks that control pattern formation is the compound eye of the fruit fly, Drosophila melanogaster. Its cellular morphogenesis has been extensively studied for more than a century and hundreds of mutants that affect its development have been isolated. In this review we will focus on the morphogenetic furrow, a wave of differentiation that takes an initially homogeneous sheet of cells and converts it into an ordered array of unit eyes or ommatidia. Since the discovery of the furrow in 1976, positive and negative acting morphogens have been thought to be solely responsible for propagating the movement of the furrow across a motionless field of cells. However, a recent study has challenged this model and instead proposed that mechanical driven cell flow also contributes to retinal pattern formation. We will discuss both models and their impact on patterning.

A morphogen is a signaling molecule that induces specific cellular responses depending on its local concentration. The concept of morphogenic gradients has been a central paradigm of developmental biology for decades. Sonic Hedgehog (Shh) is one of the most important morphogens that displays pleiotropic functions during embryonic development, ranging from neuronal patterning to axon guidance. It is commonly accepted that Shh is distributed in a gradient in several tissues from different origins during development; however, how these gradients are formed and maintained at the cellular and molecular levels is still the center of a great deal of research. In this review, we first explored all of the different sources of Shh during the development of the nervous system. Then, we detailed how these sources can distribute Shh in the surrounding tissues via a variety of mechanisms. Finally, we addressed how disrupting Shh distribution and gradients can induce severe neurodevelopmental disorders and cancers. Although the concept of gradient has been central in the field of neurodevelopment since the fifties, we also describe how contemporary leading-edge techniques, such as organoids, can revisit this classical model. [ABSTRACT FROM AUTHOR]

The Turing model (or reaction-diffusion model), first published in 1952, is a mathematical model that can account for autonomy in the morphogenesis of organisms. Although initially controversial, the model has gradually gained wider acceptance among experimental embryologists due to the accumulation of experimental data to support it. More recently, this model and others based on it have been used not only to explain biological phenomena conceptually but also as working hypotheses for molecular-level experiments and as internal components of more-complex 3D models. In this Spotlight, I will provide a personal perspective from an experimental biologist on some of the recent developments of the Turing model. [ABSTRACT FROM AUTHOR]

Morphogens determine cellular differentiation in many developing tissues in a concentration dependent manner. As a central model for gradient formation during animal development, Hedgehog (Hh) morphogens spread away from their source to direct growth and pattern formation in the Drosophila wing disc. Although heparan sulfate (HS) expression in the disc is essential for this process, it is not known whether HS regulates Hh signaling and spread in a direct or in an indirect manner. To answer this question, we systematically screened two composite Hh binding areas for HS in vitro and expressed mutated proteins in the Drosophila wing disc. We found that selectively impaired HS binding of the second site reduced Hh signaling close to the source and caused striking wing mispatterning phenotypes more distant from the source. These observations suggest that HS constrains Hh to the wing disc epithelium in a direct manner, and that interfering with this constriction converts Hh into freely diffusing forms with altered signaling ranges and impaired gradient robustness.

Neural progenitors and their neuronal progeny are bathed in extrinsic signals that impact critical decisions like the mode of cell division, how long they should reside in specific neuronal laminae, when to differentiate, and the timing of migratory decisions. Chief among these signals are secreted morphogens and extracellular matrix (ECM) molecules. Among the many cellular organelles and cell surface receptors that sense morphogen and ECM signals, the primary cilia and integrin receptors are some of the most important mediators of extracellular signals. Despite years of dissecting the function of cell-extrinsic sensory pathways in isolation, recent research has begun to show that key pathways work together to help neurons and progenitors interpret diverse inputs in their germinal niches. This mini-review utilizes the developing cerebellar granule neuron lineage as a model that highlights evolving concepts on the crosstalk between primary cilia and integrins in the development of the most abundant neuronal type in the brains of mammals.

Neural crest (NC) cells are a stem-like multipotent population of progenitor cells that are present in vertebrate embryos, traveling to various regions in the developing organism. Known as the "fourth germ layer," these cells originate in the ectoderm between the neural plate (NP), which will become the brain and spinal cord, and nonneural tissues that will become the skin and the sensory organs. NC cells can differentiate into more than 30 different derivatives in response to the appropriate signals including, but not limited to, craniofacial bone and cartilage, sensory nerves and ganglia, pigment cells, and connective tissue. The molecular and cellular mechanisms that control the induction and specification of NC cells include epigenetic control, multiple interactive and redundant transcriptional pathways, secreted signaling molecules, and adhesion molecules. NC cells are important not only because they transform into a wide variety of tissue types, but also because their ability to detach from their epithelial neighbors and migrate throughout developing embryos utilizes mechanisms similar to those used by metastatic cancer cells. In this review, we discuss the mechanisms required for the induction and specification of NC cells in various vertebrate species, focusing on the roles of early morphogenesis, cell adhesion, signaling from adjacent tissues, and the massive transcriptional network that controls the formation of these amazing cells. This article is categorized under: Nervous System Development > Vertebrates: General Principles Gene Expression and Transcriptional Hierarchies > Regulatory Mechanisms Gene Expression and Transcriptional Hierarchies > Gene Networks and Genomics Signaling Pathways > Cell Fate Signaling.

Thallusin, a highly biologically active, phytohormone-like and bacterial compound-inducing morphogenesis of the green tide-forming macroalga Ulva (Chlorophyta), was determined in bacteria and algae cultures. A sensitive and selective method was developed for quantification based on ultra-high-performance liquid chromatography coupled with electrospray ionization and a high-resolution mass spectrometer. Upon C18 solid phase extraction of the water samples, thallusin was derivatized with iodomethane to inhibit the formation of Fe–thallusin complexes interfering with the chromatographic separation. The concentration of thallusin was quantified during the relevant phases of the bacterial growth of Maribacter spp., ranging from 0.16 ± 0.01 amol cell−1 (at the peak of the exponential growth phase) to 0.86 ± 0.13 amol cell−1 (late stationary phase), indicating its accumulation in the growth medium. Finally, we directly determined the concentration of thallusin in algal culture to validate our approach for monitoring applications. Detection and quantification limits of 2.5 and 7.4 pmol L−1, respectively, were reached, which allow for quantifying ecologically relevant thallusin concentrations. Our approach will enable the surveying of thallusin in culture and in nature and will thus contribute to the chemical monitoring of aquaculture. [ABSTRACT FROM AUTHOR]

Heterocyst differentiation that occurs in some filamentous cyanobacteria, such as Anabaena sp. PCC 7120, provides a unique model for prokaryotic developmental biology. Heterocyst cells are formed in response to combined-nitrogen deprivation and possess a microoxic environment suitable for nitrogen fixation following extensive morphological and physiological reorganization. A filament of Anabaena is a true multicellular organism, as nitrogen and carbon sources are exchanged among different cells and cell types through septal junctions to ensure filament growth. Because heterocysts are terminally differentiated cells and unable to divide, their activity is an altruistic behavior dedicated to providing fixed nitrogen for neighboring vegetative cells. Heterocyst development is also a process of one-dimensional pattern formation, as heterocysts are semiregularly intercalated among vegetative cells. Morphogens form gradients along the filament and interact with each other in a fashion that fits well into the Turing model, a mathematical framework to explain biological pattern formation. [ABSTRACT FROM AUTHOR]

Heterocyst differentiation that occurs in some filamentous cyanobacteria, such as Anabaena sp. PCC 7120, provides a unique model for prokaryotic developmental biology. Heterocyst cells are formed in response to combined-nitrogen deprivation and possess a microoxic environment suitable for nitrogen fixation following extensive morphological and physiological reorganization. A filament of Anabaena is a true multicellular organism, as nitrogen and carbon sources are exchanged among different cells and cell types through septal junctions to ensure filament growth. Because heterocysts are terminally differentiated cells and unable to divide, their activity is an altruistic behavior dedicated to providing fixed nitrogen for neighboring vegetative cells. Heterocyst development is also a process of one-dimensional pattern formation, as heterocysts are semiregularly intercalated among vegetative cells. Morphogens form gradients along the filament and interact with each other in a fashion that fits well into the Turing model, a mathematical framework to explain biological pattern formation. [ABSTRACT FROM AUTHOR]

Intracellular signaling pathways are essential to establish embryonic patterning, including embryonic axis formation. Ectodermal patterning is also governed by a series of morphogens. Four ectodermal regions are thought to be controlled by morphogen gradients, but some perturbations are expected to occur during dynamic morphogenetic movement. Therefore, a mechanism to define areas precisely and reproducibly in embryos, including feedback regulation of signaling pathways, is necessary. In this review, we outline ectoderm pattern formation and signaling pathways involved in the establishment of the pre-placodal ectoderm (PPE). We also provide an example of feedback regulation of signaling pathways for robust formation of the PPE, showing the importance of this regulation. [ABSTRACT FROM AUTHOR]

The Bone Morphogenetic Protein (BMP) family reiteratively signals to direct disparate cellular fates throughout embryogenesis. In the developing dorsal spinal cord, multiple BMPs are required to specify sensory interneurons (INs). Previous studies suggested that the BMPs act as concentration-dependent morphogens to direct IN identity, analogous to the manner in which sonic hedgehog patterns the ventral spinal cord. However, it remains unresolved how multiple BMPs would cooperate to establish a unified morphogen gradient. Our studies support an alternative model: BMPs have signal-specific activities directing particular IN fates. Using chicken and mouse models, we show that the identity, not concentration, of the BMP ligand directs distinct dorsal identities. Individual BMPs promote progenitor patterning or neuronal differentiation by their activation of different type I BMP receptors and distinct modulations of the cell cycle. Together, this study shows that a 'mix and match' code of BMP signaling results in distinct classes of sensory INs.

Morphogen gradients direct the spatial patterning of developing embryos; however, the mechanisms by which these gradients are interpreted remain elusive. Here we used lattice light-sheet microscopy to perform in vivo single-molecule imaging in early Drosophila melanogaster embryos of the transcription factor Bicoid that forms a gradient and initiates patterning along the anteroposterior axis. In contrast to canonical models, we observed that Bicoid binds to DNA with a rapid off rate throughout the embryo such that its average occupancy at target loci is on-rate-dependent. We further observed Bicoid forming transient "hubs" of locally high density that facilitate binding as factor levels drop, including in the posterior, where we observed Bicoid binding despite vanishingly low protein levels. We propose that localized modulation of transcription factor on rates via clustering provides a general mechanism to facilitate binding to low-affinity targets and that this may be a prevalent feature of other developmental transcription factors.

Defining the origin of neuronal diversity is a major challenge in developmental neurobiology. The Drosophila visual system is an excellent paradigm to study how cellular diversity is generated. Photoreceptors from the eye disc grow their axons into the optic lobe and secrete Hedgehog (Hh) to induce the lamina, such that for every unit eye there is a corresponding lamina unit made up of post-mitotic precursors stacked into columns. Each differentiated column contains five lamina neuron types (L1-L5), making it the simplest neuropil in the optic lobe, yet how this diversity is generated was unknown. Here, we found that Hh pathway activity is graded along the distal-proximal axis of lamina columns, and further determined that this gradient in pathway activity arises from a gradient of Hh ligand. We manipulated Hh pathway activity cell autonomously in lamina precursors and non-cell autonomously by inactivating the Hh ligand and by knocking it down in photoreceptors. These manipulations showed that different thresholds of activity specify unique cell identities, with more proximal cell types specified in response to progressively lower Hh levels. Thus, our data establish that Hh acts as a morphogen to pattern the lamina. Although this is the first such report during Drosophila nervous system development, our work uncovers a remarkable similarity with the vertebrate neural tube, which is patterned by Sonic Hh. Altogether, we show that differentiating neurons can regulate the neuronal diversity of their distant target fields through morphogen gradients.

Secreted molecules called morphogens govern tissue patterning in a concentration-dependent manner. However, it is still unclear how reproducible patterning can be achieved with diffusing molecules, especially when that patterning concerns differentiation of thin tissues. Wnt is a morphogen that organizes cardiac development. Wnt6 patterns cardiogenic mesoderm to induce differentiation of a thin tissue, the pericardium, in Xenopus. In this study, we revealed that a Wnt receptor, frizzled-7, is expressed in a Wnt-dependent manner. With a combination of experiments and mathematical modeling, this receptor-feedback appears essential to shape a steep gradient of Wnt signaling. In addition, computer simulation revealed that this feedback imparts robustness against variations of Wnt ligand production and allows the system to reach a steady state quickly. We also found that a Wnt antagonist sFRP1, which is expressed on the opposite side of the Wnt source, accumulates on N-acetyl-rich heparan sulfate (HS). N-acetyl-rich HS concentration is high between the sources of Wnt and sFRP1, achieving local inhibition of Wnt signaling via restriction of sFRP1 spreading. These integrated regulatory systems restrict the Wnt signaling range and ensure reproducible patterning of the thin pericardium.

In many developing and regenerating systems, tissue pattern is established through gradients of informative morphogens, but we know little about how cells interpret these. Using experimental manipulation of early chick embryos, including misexpression of an inducer (VG1 or ACTIVIN) and an inhibitor (BMP4), we test two alternative models for their ability to explain how the site of primitive streak formation is positioned relative to the rest of the embryo. In one model, cells read morphogen concentrations cell-autonomously. In the other, cells sense changes in morphogen status relative to their neighbourhood. We find that only the latter model can account for the experimental results, including some counter-intuitive predictions. This mechanism (which we name the ‘neighbourhood watch’ model) illuminates the classic ‘French Flag Problem’ and how positional information is interpreted by a sheet of cells in a large developing system. [ABSTRACT FROM AUTHOR]

Germinal niche interactions and their effect on developing neurons have become the subject of intense investigation. Dissecting the complex interplay of cell-extrinsic and cell-intrinsic factors at the heart of these interactions reveals the critical basic mechanisms of neural development and how it goes awry in pediatric neurologic disorders. A full accounting of how developing neurons navigate their niches to mature and integrate into a developing neural circuit requires a combination of genetic characterization of and physical access to neurons and their supporting cell types plus transformative imaging to determine the cell biological and gene-regulatory responses to niche cues. The mouse cerebellar cortex is a prototypical experimental system meeting all of these criteria. The lessons learned therein have been scaled to other model systems and brain regions to stimulate discoveries of how developing neurons make many developmental decisions. This review focuses on how mouse cerebellar granule neuron progenitors interact with signals in their germinal niche and how that affects the neuronal differentiation and cell polarization programs that underpin lamination of the developing cerebellum. We show how modeling of these mechanisms in other systems has added to the growing evidence of how defective neuronal polarity contributes to developmental disease. [ABSTRACT FROM AUTHOR]

Experimental embryologists working at the turn of the 19th century suggested fundamental mechanisms of development, such as localized cytoplasmic determinants and tissue induction. However, the molecular basis underlying these processes proved intractable for a long time, despite concerted efforts in many developmental systems to isolate factors with a biological role. That road block was overcome by combining developmental biology with genetics. This powerful approach used unbiased genome-wide screens to isolate mutants with developmental defects and to thereby identify genes encoding key determinants and regulatory pathways that govern development. Two small invertebrates were the pioneers: the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans. Their modes of development differ in many ways, but the two together led the way to unraveling the molecular mechanisms of many fundamental developmental processes. The discovery of the grand homologies between key players in development throughout the animal kingdom underscored the usefulness of studying these small invertebrate models for animal development and even human disease. We describe developmental genetics in Drosophila and C. elegans up to the rise of genomics at the beginning of the 21st Century. Finally, we discuss themes that emerge from the histories of such distinct organisms and prospects of this approach for the future. [Display omitted] • Genome-wide mutant screens in the fly Drosophila and the worm C.elegans made major contributions to Developmental Biology. • The screens identified genes encoding key determinants and regulatory pathways that govern development. • C. elegans and Drosophila are very different invertebrate organisms with complementary strengths as models for Development. • Many regulators found in worms or flies are conserved in vertebrates and are associated with human health and disease.. [ABSTRACT FROM AUTHOR]

Germinal niche interactions and their effect on developing neurons have become the subject of intense investigation. Dissecting the complex interplay of cell-extrinsic and cell-intrinsic factors at the heart of these interactions reveals the critical basic mechanisms of neural development and how it goes awry in pediatric neurologic disorders. A full accounting of how developing neurons navigate their niches to mature and integrate into a developing neural circuit requires a combination of genetic characterization of and physical access to neurons and their supporting cell types plus transformative imaging to determine the cell biological and gene-regulatory responses to niche cues. The mouse cerebellar cortex is a prototypical experimental system meeting all of these criteria. The lessons learned therein have been scaled to other model systems and brain regions to stimulate discoveries of how developing neurons make many developmental decisions. This review focuses on how mouse cerebellar granule neuron progenitors interact with signals in their germinal niche and how that affects the neuronal differentiation and cell polarization programs that underpin lamination of the developing cerebellum. We show how modeling of these mechanisms in other systems has added to the growing evidence of how defective neuronal polarity contributes to developmental disease.

For over 40 years, the Bicoid-hunchback (Bcd-hb) system in the fruit fly embryo has been used as a model to study how positional information in morphogen concentration gradients is robustly translated into step-like responses. A body of quantitative comparisons between theory and experiment have since questioned the initial paradigm that the sharp hb transcription pattern emerges solely from diffusive biochemical interactions between the Bicoid transcription factor and the gene promoter region. Several alternative mechanisms have been proposed, such as additional sources of positional information, positive feedback from Hb proteins or out-of-equilibrium transcription activation. By using the MS2-MCP RNA-tagging system and analysing in real time, the transcription dynamics of synthetic reporters for Bicoid and/or its two partners Zelda and Hunchback, we show that all the early hb expression pattern features and temporal dynamics are compatible with an equilibrium model with a short decay length Bicoid activity gradient as a sole source of positional information. Meanwhile, Bicoid’s partners speed-up the process by different means: Zelda lowers the Bicoid concentration threshold required for transcriptional activation while Hunchback reduces burstiness and increases the polymerase firing rate.

Successful development depends on the creation of spatial gradients of transcription factors within developing fields, and images of graded distributions of gene products populate the pages of developmental biology journals. Therefore the challenge is to understand how the graded levels of intracellular transcription factors are generated across fields of cells. We propose that transcription factor gradients are generated as a result of an underlying gradient of cell cycle lengths. Very long cell cycles will permit accumulation of a high level of a gene product encoded by a large transcription unit, whereas shorter cell cycles will permit progressively fewer transcripts to be completed due to gating of transcription by the cell cycle. We also propose that the gradients of cell cycle lengths are generated by gradients of extracellular morphogens/growth factors. The model of cell cycle gated transcriptional regulation brings focus back to the functional role of morphogens as cell cycle regulators, and proposes a specific and testable mechanism by which morphogens, in their roles as growth factors (how they were originally discovered), also determine cell fate.

Morphogen gradients induce sharply defined domains of gene expression in a concentration-dependent manner, yet how cells interpret these signals in the face of spatial and temporal noise remains unclear. Using fluorescence lifetime imaging microscopy (FLIM) and phasor analysis to measure endogenous retinoic acid (RA) directly in vivo, we have investigated the amplitude of noise in RA signaling, and how modulation of this noise affects patterning of hindbrain segments (rhombomeres) in the zebrafish embryo. We demonstrate that RA forms a noisy gradient during critical stages of hindbrain patterning and that cells use distinct intracellular binding proteins to attenuate noise in RA levels. Increasing noise disrupts sharpening of rhombomere boundaries and proper patterning of the hindbrain. These findings reveal novel cellular mechanisms of noise regulation, which are likely to play important roles in other aspects of physiology and disease.

The ancestral Chordin/bone morphogenetic protein (BMP) signaling pathway that establishes dorsal-ventral (D-V) patterning in animal development is one of the best understood morphogenetic gradients, and is established by multiple proteins that interact with each other in the extracellular space-including several BMPs, Chordin, Tolloid, Ont-1, Crossveinless-2, and Sizzled. The D-V gradient is adjusted redundantly by regulating the synthesis of its components, by direct protein-protein interactions between morphogens, and by long-range diffusion. The entire embryo participates in maintaining the D-V BMP gradient, so that for each action in the dorsal side there is a reaction in the ventral side. A gradient of Chordin is formed in the extracellular matrix that separates ectoderm from endomesoderm, called Brachet's cleft in Xenopus. The Chordin/BMP pathway is self-organizing and able to scale pattern in the dorsal half of bisected embryos or in Spemann dorsal lip transplantation experiments.

Morphogens were originally defined as secreted signaling molecules that diffuse from local sources to form concentration gradients, which specify multiple cell fates. More recently morphogen gradients have been shown to incorporate a range of mechanisms including short-range signal activation, transcriptional/translational feedback, and temporal windows of target gene induction. Many critical cell-cell signals implicated in both embryonic development and disease, such as Wnt, fibroblast growth factor (Fgf), hedgehog (Hh), transforming growth factor beta (TGFb), and retinoic acid (RA), are thought to act as morphogens, but key information on signal propagation and ligand distribution has been lacking for most. The zebrafish provides unique advantages for genetics and imaging to address gradients during early embryonic stages when morphogens help establish major body axes. This has been particularly informative for RA, where RA response elements (RAREs) driving fluorescent reporters as well as Fluorescence Resonance Energy Transfer (FRET) reporters of receptor binding have provided evidence for gradients, as well as regulatory mechanisms that attenuate noise and enhance gradient robustness in vivo. Here we summarize available tools in zebrafish and discuss their utility for studying dynamic regulation of RA morphogen gradients, through combined experimental and computational approaches.

The ancestral Chordin/bone morphogenetic protein (BMP) signaling pathway that establishes dorsal-ventral (D-V) patterning in animal development is one of the best understood morphogenetic gradients, and is established by multiple proteins that interact with each other in the extracellular space-including several BMPs, Chordin, Tolloid, Ont-1, Crossveinless-2, and Sizzled. The D-V gradient is adjusted redundantly by regulating the synthesis of its components, by direct protein-protein interactions between morphogens, and by long-range diffusion. The entire embryo participates in maintaining the D-V BMP gradient, so that for each action in the dorsal side there is a reaction in the ventral side. A gradient of Chordin is formed in the extracellular matrix that separates ectoderm from endomesoderm, called Brachet's cleft in Xenopus. The Chordin/BMP pathway is self-organizing and able to scale pattern in the dorsal half of bisected embryos or in Spemann dorsal lip transplantation experiments.

Morphogen gradients are known to subdivide a naive cell field into distinct zones of gene expression. Here, we examine whether morphogens can also induce a graded response within such domains. To this end, we explore the role of the Dorsal protein nuclear gradient along the dorsoventral axis in defining the graded pattern of actomyosin constriction that initiates gastrulation in early Drosophila embryos. Two complementary mechanisms for graded accumulation of mRNAs of crucial zygotic Dorsal target genes were identified. First, activation of target-gene expression expands over time from the ventral-most region of high nuclear Dorsal to lateral regions, where the levels are lower, as a result of a Dorsal-dependent activation probability of transcription sites. Thus, sites that are activated earlier will exhibit more mRNA accumulation. Second, once the sites are activated, the rate of RNA Polymerase II loading is also dependent on Dorsal levels. Morphological restrictions require that translation of the graded mRNA be delayed until completion of embryonic cell formation. Such timing is achieved by large introns, which provide a delay in production of the mature mRNAs. Spatiotemporal regulation of key zygotic genes therefore shapes the pattern of gastrulation. [ABSTRACT FROM AUTHOR]

To study the mechanisms underlying developmental pattern formation in a tissue, one needs to analyze the dynamics of the regulators in time and space across the tissue of a specific architecture. This problem is essential for the developmental regulators (morphogens) that distribute over the tissues anisotropically, forming there maxima and gradients and guiding cellular processes in a dose-dependent manner. Here we present the PlantLayout pipeline for MATLAB software, which facilitates the computational studies of tissue patterning. With its help, one can build a structural model of a two-dimensional tissue, embed it into a mathematical model in ODEs, perform numerical simulations, and visualize the obtained results - everything on the same platform. As a result, one can study the concentration dynamics of developmental regulators over the cell layout reconstructed from the real tissue. PlantLayout allows studying the dynamics and the output of gene networks guided by the developmental regulator in specific cells. The gene networks could be different for different cell types. One of the obstacles that PlantLayout removes semi-automatically is the determination of the cell wall orientation which is relevant when cells in the tissue have a polarity. Additionally, PlantLayout allows automatically extracting other quantitative and qualitative features of the cells and the cell walls, which might help in the modeling of a developmental pattern, such as the length and the width of the cell walls, the set of the neighboring cells, cell volume and cell perimeter. We demonstrate PlantLayout performance on the model of phytohormone auxin distribution over the plant root tip.