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Much of the striking diversity of life on Earth has arisen from variations in the way that the same molecules and networks operate during development to shape and pattern tissues and organs into different morphologies. However, we still understand very little about the potential for diversification exhibited by different, highly conserved mechanisms during evolution, or, conversely, the constraints that they place on evolution. With the aim of steering the field in new directions, we focus on morphogen-mediated patterning and growth as a case study to demonstrate how conserved developmental mechanisms can adapt during evolution to drive morphological diversification and optimise functionality, and to illustrate how evolution algorithms and computational tools can be used alongside experiments to provide insights into how these conserved mechanisms can evolve. We first introduce key conserved properties of morphogen-driven patterning mechanisms, before summarising comparative studies that exemplify how changes in the spatiotemporal expression and signalling levels of morphogens impact the diversification of organ size, shape and patterning in nature. Finally, we detail how theoretical frameworks can be used in conjunction with experiments to probe the role of morphogen driven patterning mechanisms in evolution. We conclude that morphogen-mediated patterning is an excellent model system and offers a generally applicable framework to investigate the evolution of developmental mechanisms. [ABSTRACT FROM AUTHOR]

Adult planarians can grow when fed and degrow (shrink) when starved while maintaining their whole-body shape. It is unknown how the morphogens patterning the planarian axes are coordinated during feeding and starvation or how they modulate the necessary differential tissue growth or degrowth. Here, we investigate the dynamics of planarian shape together with a theoretical study of the mechanisms regulating whole-body proportions and shape. We found that the planarian body proportions scale isometrically following similar linear rates during growth and degrowth, but that fed worms are significantly wider than starved worms. By combining a descriptive model of planarian shape and size with a mechanistic model of anterior-posterior and medio-lateral signaling calibrated with a novel parameter optimization methodology, we theoretically demonstrate that the feedback loop between these positional information signals and the shape they control can regulate the planarian whole-body shape during growth. Furthermore, the computational model produced the correct shape and size dynamics during degrowth as a result of a predicted increase in apoptosis rate and pole signal during starvation. These results offer mechanistic insights into the dynamic regulation of whole-body morphologies. [ABSTRACT FROM AUTHOR]

Neural crest cells (NCCs) are a dynamic, multipotent, vertebrate-specific population of embryonic stem cells. These ectodermally-derived cells contribute to diverse tissue types in developing embryos including craniofacial bone and cartilage, the peripheral and enteric nervous systems and pigment cells, among a host of other cell types. Due to their contribution to a significant number of adult tissue types, the mechanisms that drive their formation, migration and differentiation are highly studied. NCCs have a unique ability to transition from tightly adherent epithelial cells to mesenchymal and migratory cells by altering their polarity, expression of cell-cell adhesion molecules and gaining invasive abilities. In this Review, we discuss classical and emerging factors driving NCC epithelial-to-mesenchymal transition and migration, highlighting the role of signaling and transcription factors, as well as novel modifying factors including chromatin remodelers, small RNAs and post-translational regulators, which control the availability and longevity of major NCC players.

Morphogen gradients provide essential positional information to gene networks through their spatially heterogeneous distribution, yet how they form is still hotly contested, with multiple models proposed for different systems. Here, we focus on the transcription factor Bicoid (Bcd), a morphogen that forms an exponential gradient across the anterior-posterior (AP) axis of the early Drosophila embryo. Using fluorescence correlation spectroscopy we find there are spatial differences in Bcd diffusivity along the AP axis, with Bcd diffusing more rapidly in the posterior. We establish that such spatially varying differences in Bcd dynamics are sufficient to explain how Bcd can have a steep exponential gradient in the anterior half of the embryo and yet still have an observable fraction of Bcd near the posterior pole. In the nucleus, we demonstrate that Bcd dynamics are impacted by binding to DNA. Addition of the Bcd homeodomain to eGFP::NLS qualitatively replicates the Bcd concentration profile, suggesting this domain regulates Bcd dynamics. Our results reveal how a long-range gradient can form while retaining a steep profile through much of its range. [ABSTRACT FROM AUTHOR]

Butterfly color patterns provide visible and biodiverse phenotypic readouts of the patterning processes. Although the secreted ligand WntA has been shown to instruct the color pattern formation in butterflies, its mode of reception remains elusive. Butterfly genomes encode four homologs of the Frizzled-family of Wnt receptors. Here, we show that CRISPR mosaic knockouts of frizzled2 (fz2) phenocopy the color pattern effects of WntA loss of function in multiple nymphalids. Whereas WntA mosaic clones result in intermediate patterns of reduced size, fz2 clones are cellautonomous, consistent with a morphogen function. Shifts in expression of WntA and fz2 in WntA crispant pupae show that they are under positive and negative feedback, respectively. Fz1 is required for Wnt-independent planar cell polarity in the wing epithelium. Fz3 and Fz4 show phenotypes consistent with Wnt competitive-antagonist functions in vein formation (Fz3 and Fz4), wing margin specification (Fz3), and color patterning in the Discalis and Marginal Band Systems (Fz4). Overall, these data show that the WntA/Frizzled2 morphogen-receptor pair forms a signaling axis that instructs butterfly color patterning and shed light on the functional diversity of insect Frizzled receptors. [ABSTRACT FROM AUTHOR]

Limb regeneration is the outcome of a complex sequence of events that are mediated by interactions between cells derived from the tissues of the amputated stump. Early in regeneration, these interactions are mediated by growth factor/morphogen signaling associated with nerves and the wound epithelium. One shared property of these proregenerative signaling molecules is that their activity is dependent on interactions with sulfated glycosaminoglycans (GAGs), heparan sulfate proteoglycan (HSPG) in particular, in the extracellular matrix (ECM). We hypothesized that there are cells in the axolotl that synthesize specific HSPGs that control growth factor signaling in time and space. In this study we have identified a subpopulation of cells within the ECM of axolotl skin that express high levels of sulfated GAGs on their cell surface. These cells are dispersed in a grid-like pattern throughout the dermis as well as the loose connective tissues that surround the tissues of the limb. These cells alter their morphology during regeneration, and are candidates for being a subpopulation of connective tissue cells that function as the cells required for pattern-formation during regeneration. Given their high level of HSPG expression, their stellate morphology, and their distribution throughout the loose connective tissues, we refer to these as the positional information GRID (Groups that are Regenerative, Interspersed and Dendritic) cells. In addition, we have identified cells that stain for high levels of expression of sulfated GAGs in mouse limb connective tissue that could have an equivalent function to GRID cells in the axolotl. The identification of GRID cells may have important implications for work in the area of Regenerative Engineering.

The formation of identical twins from a single egg has fascinated developmental biologists for a very long time. Previous work had shown that Xenopus blastulae bisected along the dorsal-ventral (D-V) midline (i.e., the sagittal plane) could generate twins but at very low frequencies. Here, we have improved this method by using an eyelash knife and changing saline solutions, reaching frequencies of twinning of 50% or more. This allowed mechanistic analysis of the twinning process. We unexpectedly observed that the epidermis of the resulting twins was asymmetrically pigmented at the tailbud stage of regenerating tadpoles. This pigment was entirely of maternal (oocyte) origin. Bisecting the embryo generated a large wound, which closed from all directions within 60 minutes, bringing cells normally fated to become Spemann organizer in direct contact with predicted ventral-most cells. Lineage-tracing analyses at the four-cell stage showed that in regenerating embryos midline tissues originated from the dorsal half, while the epidermis was entirely of ventral origin. Labeling of D-V segments at the 16-cell stage showed that the more pigmented epidermis originated from the ventral-most cells, while the less-pigmented epidermis arose from the adjoining ventral segment. This suggested a displacement of the organizer by 90°. Studies with the marker Chordin and phospho-Smad1/5/8 showed that in half embryos a new D-V gradient is intercalated at the site of the missing half. The displacement of self-organizing morphogen gradients uncovered here may help us understand not only twin formation in amphibians, but also rare cases of polyembryony.

Morphogens of the Hh family trigger gene expression changes in receiving cells in a concentration-dependent manner to regulate their identity, proliferation, death or metabolism, depending on the tissue or organ. This variety of responses relies on a conserved signaling pathway. Its logic includes a negative-feedback loop involving the Hh receptor Ptc. Here, using experiments and computational models we study and compare the different spatial signaling profiles downstream of Hh in several developing Drosophila organs. We show that the spatial distributions of Ptc and the activator transcription factor CiA in wing, antenna and ocellus show similar features, but are markedly different from that in the compound eye. We propose that these two profile types represent two time points along the signaling dynamics, and that the interplay between the spatial displacement of the Hh source in the compound eye and the negative-feedback loop maintains the receiving cells effectively in an earlier stage of signaling. These results show how the interaction between spatial and temporal dynamics of signaling and differentiation processes may contribute to the informational versatility of the conserved Hh signaling pathway. [ABSTRACT FROM AUTHOR]

The recent literature on causation has seen the introduction of several distinctions within causation, which are thought to be important for understanding the widespread scientific practice of focusing causal explanations on a subset of the factors that are causally relevant for a phenomenon. Concepts used to draw such distinctions include, among others, stability, specificity, proportionality, or actual-difference making. In this contribution, I propose a new distinction that picks out an explanatorily salient class of causes in biological systems. Some select causes in complex biological systems, I argue, have the property of enabling coherent causal control of these systems. Examples of such control variables include hormones and other signaling molecules, e.g., TOR (target of rapamycin), morphogens or the products of homeotic selector genes in embryonic pattern formation. I propose an analysis of this notion based on concepts borrowed from causal graph theory. [ABSTRACT FROM AUTHOR]

This contribution considers proto-cell structures associated with asymmetries, mainly gravity, in the framework of reaction–diffusion. There are equivalent solutions for defined morphogen parameters in the equations that allow for defining proto-tissue complexity and configurational entropy. Using RNA data, improvements to the complexity and entropy due to the Earth's gravity are presented. The theoretical proto-tissues complexity estimation, as a function of arbitrary surface gravity, is likewise proposed. In this sense, hypothetical aggregates of proto-cells on Mars would have a lower complexity than on Earth, which is equally valid for the Moon. Massive planets, or exoplanets like BD+20594b, could have major proto-tissue complexity and, eventually, rich biodiversity. [ABSTRACT FROM AUTHOR]

Morphogens choreograph the generation of remarkable cellular diversity in the developing nervous system. Differentiation of stem cells in vitro often relies upon the combinatorial modulation of these signaling pathways. However, the lack of a systematic approach to understand morphogen-directed differentiation has precluded the generation of many neural cell populations, and the general principles of regional specification and maturation remain incomplete. Here, we developed an arrayed screen of 14 morphogen modulators in human neural organoids cultured for over 70 days. Deconvolution of single-cell-multiplexed RNA sequencing data revealed design principles of brain region specification. We tuned neural subtype diversity to generate a tachykinin 3 (TAC3)-expressing striatal interneuron type within assembloids. To circumvent limitations of in vitro neuronal maturation, we used a neonatal rat transplantation strategy that enabled human Purkinje neurons to develop their hallmark complex dendritic branching. This comprehensive platform yields insights into the factors influencing stem cell-derived neural diversification and maturation., Competing Interests: Declaration of interests G.N. was an employee of Daiichi-Sankyo Co., Ltd, when performing the experiments for this study; however, the company did not have any input and interpretation on the design of experiments and the data. Stanford University holds patents that cover the generation of regionalized neural organoids., (Copyright © 2024 Elsevier Inc. All rights reserved.)

As single cells, ciliates build, duplicate, and even regenerate complex cortical patterns by largely unknown mechanisms that precisely position organelles along two cell‐wide axes: anterior–posterior and circumferential (left–right). We review our current understanding of intracellular patterning along the anterior–posterior axis in ciliates, with emphasis on how the new pattern emerges during cell division. We focus on the recent progress at the molecular level that has been driven by the discovery of genes whose mutations cause organelle positioning defects in the model ciliate Tetrahymena thermophila. These investigations have revealed a network of highly conserved kinases that are confined to either anterior or posterior domains in the cell cortex. These pattern‐regulating kinases create zones of cortical inhibition that by exclusion determine the precise placement of organelles. We discuss observations and models derived from classical microsurgical experiments in large ciliates (including Stentor) and interpret them in light of recent molecular findings in Tetrahymena. In particular, we address the involvement of intracellular gradients as vehicles for positioning organelles along the anterior‐posterior axis. [ABSTRACT FROM AUTHOR]

The sizes of living organisms range over twenty orders of magnitude. Within the same species, the size of individuals also varies according to the environmental conditions to which they are subjected. From the studies conducted on organisms as diverse as the drosophila, the salamander or the mouse, laws and conserved mechanisms emerge that shed light on the fundamental aspects of growth, but also on more medical issues such as tissue regeneration, metabolic homeostasis and cancer.

Abstract Background Organisms show an incredibly diverse array of body and organ shapes that are both unique to their taxon and important for adapting to their environment. Achieving these specific shapes involves coordinating the many processes that transform single cells into complex organs, and regulating their growth so that they can function within a fully-formed body. Main text Conceptually, body and organ shape can be separated in two categories, although in practice these categories need not be mutually exclusive. Body shape results from the extent to which organs, or parts of organs, grow relative to each other. The patterns of relative organ size are characterized using allometry. Organ shape, on the other hand, is defined as the geometric features of an organ’s component parts excluding its size. Characterization of organ shape is frequently described by the relative position of homologous features, known as landmarks, distributed throughout the organ. These descriptions fall into the domain of geometric morphometrics. Conclusion In this review, we discuss the methods of characterizing body and organ shape, the developmental programs thought to underlie each, highlight when and how the mechanisms regulating body and organ shape might overlap, and provide our perspective on future avenues of research.

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

Organ and tissue growth result from an integration of biophysical communication across biological scales, both in time and space. In this review, we highlight new insight into the dynamic connections between control mechanisms operating at different length scales. First, we consider how the dynamics of chemical and electrical signaling in the shape of gradients or waves affect spatiotemporal signal interpretation. Then, we discuss the mechanics underlying dynamic cell behavior during oriented tissue growth, followed by the connections between signaling at the tissue and organismal levels. [Display omitted] [ABSTRACT FROM AUTHOR]

This contribution considers proto-cell structures associated with asymmetries, mainly gravity, in the framework of reaction–diffusion. There are equivalent solutions for defined morphogen parameters in the equations that allow for defining proto-tissue complexity and configurational entropy. Using RNA data, improvements to the complexity and entropy due to the Earth’s gravity are presented. The theoretical proto-tissues complexity estimation, as a function of arbitrary surface gravity, is likewise proposed. In this sense, hypothetical aggregates of proto-cells on Mars would have a lower complexity than on Earth, which is equally valid for the Moon. Massive planets, or exoplanets like BD+20594b, could have major proto-tissue complexity and, eventually, rich biodiversity.

Chloride intracellular channel 4 (CLIC4) is a recently discovered driver of fibroblast activation in Scleroderma (SSc) and cancer-associated fibroblasts (CAF). CLIC4 expression and activity are regulated by TGF-β signalling through the SMAD3 transcription factor. In view of the aberrant activation of canonical Wnt-3a and Hedgehog (Hh) signalling in fibrosis, we investigated their role in CLIC4 upregulation. Here, we show that TGF-β/SMAD3 co-operates with Wnt3a/β-catenin and Smoothened/GLI signalling to drive CLIC4 expression in normal dermal fibroblasts, and that the inhibition of β-catenin and GLI expression or activity abolishes TGF-β/SMAD3-dependent CLIC4 induction. We further show that the expression of the pro-fibrotic marker α-smooth muscle actin strongly correlates with CLIC4 expression in dermal fibroblasts. Further investigations revealed that the inhibition of CLIC4 reverses morphogen-dependent fibroblast activation. Our data highlights that CLIC4 is a common downstream target of TGF-β, Hh, and Wnt-3a through signalling crosstalk and we propose a potential therapeutic avenue using CLIC4 inhibitors

Members of the Hedgehog family of morphogens mediate the intercellular communication necessary for the organisation and development of many animal tissues. They are modified by various lipid adducts, rendering them insoluble in hydrophilic environments and leading to the contentious question of how these molecules travel in the aqueous extracellular space. Seminal work carried out by Suzanne Eaton and her colleagues has shed light on how these morphogens can spread over long distances through their association with lipoprotein particles. In this Spotlight article,we discuss Suzanne's pioneering work and her contribution to our understanding of the transport and activity of morphogens, in particular Hedgehog.We also describe two other essential aspects of her work: the discovery and characterisation of endogenously present Hedgehog variants, as well as her proposition that, in addition to its role as a morphogen, Hedgehog acts as an endocrine hormone. [ABSTRACT FROM AUTHOR]

Recent advances in stem-cell technologies include the differentiation of human embryonic stem cells (hESCs) into organ-like structures (organoids). These organoids exhibit remarkable self-organization that resembles key aspects of in vivo organ development. However, organoids have an unpredictable anatomy, and poorly reflect the topography of the dorsoventral, mediolateral, and anteroposterior axes. In vivo the temporal and the spatial patterning of the developing tissue is orchestrated by signaling molecules called morphogens. Here, we used morphogen-soaked beads to influence the spatial identities within hESC-derived brain organoids. The morphogen- and synthetic molecules-soaked beads were interpreted as local organizers, and key transcription factor expression levels within the organoids were affected as a function of the distance fromthe bead. We used an on-chip imaging device that we have developed, that allows live imaging of the developing hESC-derived organoids. This platform enabled studying the effect of changes in WNT/BMP gradients on the expression of key landmark genes in the on-chip human brain organoids. Titration of CHIR99201 (WNT agonist) and BMP4 directed the expression of telencephalon and medial pallium genes; dorsal and ventral midbrain markers; and isthmus-related genes. Overall, our protocol provides an opportunity to study phenotypes of altered regional specification and defected connectivity, which are found in neurodevelopmental diseases. [ABSTRACT FROM AUTHOR]

Periodic patterning is widespread in development and can be modelled by reaction-diffusion (RD) processes.However, minimal two-component RD descriptions are vastly simpler than the multi-molecular events that actually occur and are often hard to relate to real interactions measured experimentally. Addressing these issues, we investigated the periodic striped patterning of the rugae (transverse ridges) in the mammalian oral palate, focusing on multiple previously implicated pathways: FGF, Hh, Wnt and BMP. For each, we experimentally identified spatial patterns of activity and distinct responses of the system to inhibition. Through numerical and analytical approaches, we were able to constrain substantially the number of network structures consistent with the data. Determination of the dynamics of pattern appearance further revealed its initiation by 'activators' FGF and Wnt, and 'inhibitor' Hh, whereas BMP and mesenchyme-specific-FGF signalling were incorporated once stripes were formed. This further limited the number of possible networks. Experimental constraint thus limited the number of possibleminimal networks to 154, just 0.004% of the number of possible diffusion-driven instability networks. Together, these studies articulate the principles of multi-morphogen RD patterning and demonstrate the utility of perturbation analysis for constraining RD systems. [ABSTRACT FROM AUTHOR]

The genetic network controlling early dorsal-ventral (DV) patterning has been extensively studied and modeled in the fruit fly Drosophila. This patterning is driven by signals coming from bone morphogenetic proteins (BMPs), and regulated by interactions of BMPs with secreted factors such as the antagonist short gastrulation (Sog). Experimental studies suggest that the DV patterning of vertebrates is controlled by a similar network of BMPs and antagonists (such as Chordin, a homologue of Sog), but differences exist in how the two systems are organized, and a quantitative comparison of pattern formation in them has not been made. Here, we develop a computational model in three dimensions of the zebrafish embryo and use it to study molecular interactions in the formation of BMP morphogen gradients in early DV patterning. Simulation results are presented on the dynamics BMP gradient formation, the cooperative action of two feedback loops from BMP signaling to BMP and Chordin synthesis, and pattern sensitivity with respect to BMP and Chordin dosage. Computational analysis shows that, unlike the case in Drosophila, synergy of the two feedback loops in the zygotic control of BMP and Chordin expression, along with early initiation of localized Chordin expression, is critical for establishment and maintenance of a stable and appropriate BMP gradient in the zebrafish embryo.

A previously investigated basic model (System B) for the study of signaling morphogen gradient formation that allows for reversible binding of morphogens (aka ligands) with signaling receptors, degradation of bound morphogens and diffusion of unbound morphogens is extended to include the effects of membrane-bound non-signaling molecules (or non-receptors for short) such as proteoglycans that bind reversibly with the same morphogens and degrade them. Our main goal is to delineate the effects of the presence of non-receptors on the existence and properties of the steady-state concentration gradient of signaling ligand-receptor complexes. Stability of the steady-state morphogen gradients is established and the time to reach steady-state behavior after the onset of morphogen production will be analyzed. The theoretical findings offer explanations for observations reported in several previous experiments on Drosophila wing imaginal discs.

Recent reports have documented the differentiation of human pluripotent stem cells toward the skeletal myogenic lineage using transgene- and cell purification-free approaches. Although these protocols generate myocytes, they have not demonstrated scalability, safety, and in vivo engraftment, which are key aspects for their future clinical application. Here we recapitulate one prominent protocol, and show that it gives rise to a heterogeneous cell population containing myocytes and other cell types. Upon transplantation, the majority of human donor cells could not contribute to myofiber formation. As a proof-of-principle, we incorporated the inducible PAX7 lentiviral system into this protocol, which then enabled scalable expansion of a homogeneous population of skeletal myogenic progenitors capable of forming myofibers in vivo. Our findings demonstrate the methods for scalable expansion of PAX7+ myogenic progenitors and their purification are critical for practical application to cell replacement treatment of muscle degenerative diseases.

Human brain development involves an orchestrated, massive neural progenitor expansion while a multi-cellular tissue architecture is established. Continuously expanding organoids can be grown directly from multiple somatic tissues, yet to date, brain organoids can solely be established from pluripotent stem cells. Here, we show that healthy human fetal brain in vitro self-organizes into organoids (FeBOs), phenocopying aspects of in vivo cellular heterogeneity and complex organization. FeBOs can be expanded over long time periods. FeBO growth requires maintenance of tissue integrity, which ensures production of a tissue-like extracellular matrix (ECM) niche, ultimately endowing FeBO expansion. FeBO lines derived from different areas of the central nervous system (CNS), including dorsal and ventral forebrain, preserve their regional identity and allow to probe aspects of positional identity. Using CRISPR-Cas9, we showcase the generation of syngeneic mutant FeBO lines for the study of brain cancer. Taken together, FeBOs constitute a complementary CNS organoid platform., Competing Interests: Declaration of interests D.H., B.A., and H.C. are inventors on a patent related to this work. The full disclosure of H.C. is given at https://www.uu.nl/staff/JCClevers/., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

In order to contribute to the appropriate tissues during development, cells need to know their position within the embryo. This positional information is conveyed by gradients of signaling molecules, termed morphogens, that are produced in specific regions of the embryo and induce concentration-dependent responses in target tissues. Positional information is remarkably robust, and embryos often develop with the correct proportions even if large parts of the embryo are removed. In this Review, we discuss classical embryological experiments and modern quantitative analyses that have led to mechanistic insights into how morphogen gradients adapt, scale and properly pattern differently sized domains. We analyze these experimental findings in the context of mathematical models and synthesize general principles that apply to multiple systems across species and developmental stages. [ABSTRACT FROM AUTHOR]

Neural induction and patterning in vertebrates are regulated during early development by several morphogens, such as bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs). Ventral ectoderm differentiates into epidermis in response to BMPs, whereas BMP signaling is tightly inhibited in the dorsal ectoderm which develops into neural tissues. Here, we show that Cdc2‐like kinase 2 (Clk2) promotes early neural development and inhibits epidermis differentiation in Xenopus embryos. clk2 is specifically expressed in neural tissues along the anterior‐posterior axis during early Xenopus embryogenesis. When overexpressed in ectodermal explants, Clk2 induces the expression of both anterior and posterior neural marker genes. In agreement with this observation, overexpression of Clk2 in whole embryos expands the neural plate at the expense of epidermal ectoderm. Interestingly, the neural‐inducing activity of Clk2 is increased following BMP inhibition and activation of the FGF signaling pathway in ectodermal explants. Clk2 also downregulates the level of p‐Smad1/5/8 in cooperation with BMP inhibition, in addition to increasing the level of activated MAPK together with FGF. These results suggest that Clk2 plays a role in early neural development of Xenopus possibly via modulation of morphogen signals such as the BMP and FGF pathways. [ABSTRACT FROM AUTHOR]

Developmental system drift is a likely mechanism for the origin of hybrid incompatibilities between closely related species. We examine here the detailed mechanistic basis of hybrid incompatibilities between two allopatric lineages, for a genotype-phenotype map of developmental system drift under stabilising selection, where an organismal phenotype is conserved, but the underlying molecular phenotypes and genotype can drift. This leads to number of emergent phenomenon not obtainable by modelling genotype or phenotype alone. Our results show that: 1) speciation is more rapid at smaller population sizes with a characteristic, Orr-like, power law, but at large population sizes slow, characterised by a sub-diffusive growth law; 2) the molecular phenotypes under weakest selection contribute to the earliest incompatibilities; and 3) pair-wise incompatibilities dominate over higher order, contrary to previous predictions that the latter should dominate. The population size effect we find is consistent with previous results on allopatric divergence of transcription factor-DNA binding, where smaller populations have common ancestors with a larger drift load because genetic drift favours phenotypes which have a larger number of genotypes (higher sequence entropy) over more fit phenotypes which have far fewer genotypes; this means less substitutions are required in either lineage before incompatibilities arise. Overall, our results indicate that biophysics and population size provide a much stronger constraint to speciation than suggested by previous models, and point to a general mechanistic principle of how incompatibilities arise the under stabilising selection for an organismal phenotype. [ABSTRACT FROM AUTHOR]

The article reports that Wnts are secreted, lipidated glycoproteins that are essential for cell–cell communication in development and tissue homeostasis throughout metazoans. The W in Wnt comes from the wingless (wg) gene, which is required for proper formation of most tissues and appendages in Drosophila.

The complexity of morphogenesis poses a fundamental challenge to understanding the mechanisms governing the formation of biological patterns and structures. Over the past century, numerous processes have been identified as critically contributing to morphogenetic events, but the interplay between the various components and aspects of pattern formation have been much harder to grasp. The combination of traditional biology with mathematical and computational methods has had a profound effect on our current understanding of morphogenesis and led to significant insights and advancements in the field. In particular, the theoretical concepts of reaction–diffusion systems and positional information, proposed by Alan Turing and Lewis Wolpert, respectively, dramatically influenced our general view of morphogenesis, although typically in isolation from one another. In recent years, agent-based modeling has been emerging as a consolidation and implementation of the two theories within a single framework. Agent-based models (ABMs) are unique in their ability to integrate combinations of heterogeneous processes and investigate their respective dynamics, especially in the context of spatial phenomena. In this review, we highlight the benefits and technical challenges associated with ABMs as tools for examining morphogenetic events. These models display unparalleled flexibility for studying various morphogenetic phenomena at multiple levels and have the important advantage of informing future experimental work, including the targeted engineering of tissues and organs. [ABSTRACT FROM AUTHOR]

WNT/β-catenin signaling is crucial to all stages of life. It controls early morphogenetic events in embryos, maintains stem cell niches in adults, and is dysregulated in many types of cancer. Despite its ubiquity, little is known about the dynamics of signal transduction or whether it varies across contexts. Here we probe the dynamics of signaling by monitoring nuclear accumulation of β-catenin, the primary transducer of canonical WNT signals, using quantitative live cell imaging. We show that β-catenin signaling responds adaptively to constant WNT signaling in pluripotent stem cells, and that these dynamics become sustained on differentiation. Varying dynamics were also observed in the response to WNT in commonly used mammalian cell lines. Signal attenuation in pluripotent cells is observed even at saturating doses, where ligand stability does not affect the dynamics. TGFβ superfamily ligands Activin and BMP, which coordinate with WNT signaling to pattern the gastrula, increase the β-catenin response in a manner independent of their ability to induce new WNT ligand production. Our results reveal how variables external to the pathway, including differentiation status and cross-talk with other pathways, dramatically alter WNT/β-catenin dynamics. [ABSTRACT FROM AUTHOR]

Pollen provides an excellent system to study pattern formation at the single-cell level. Pollen surface is covered by the pollen wall exine, whose deposition is excluded from certain surface areas, the apertures, which vary between the species in their numbers, positions, and morphology. What determines aperture patterns is not understood. Arabidopsis thaliana normally develops three apertures, equally spaced along the pollen equator. However, Arabidopsis mutants whose pollen has higher ploidy and larger volume develop four or more apertures. To explore possible mechanisms responsible for aperture patterning, we developed a mathematical model based on the Gierer-Meinhardt system of equations. This model was able to recapitulate aperture patterns observed in the wild-type and higher-ploidy pollen. We then used this model to further explore geometric and kinetic factors that may influence aperture patterns and found that pollen size, as well as certain kinetic parameters, like diffusion and decay of morphogens, could play a role in formation of aperture patterns. In conjunction with mathematical modeling, we also performed a forward genetic screen in Arabidopsis and discovered two mutants with aperture patterns that had not been previously observed in this species but were predicted by our model. The macaron mutant develops a single ring-like aperture, matching the unusual ring-like pattern produced by the model. The doughnut mutant forms two pore-like apertures at the poles of the pollen grain. Further tests on these novel mutants, motivated by the modeling results, suggested the existence of an area of inhibition around apertures that prevents formation of additional apertures in their vicinity. This work demonstrates the ability of the theoretical model to help focus experimental efforts and to provide fundamental insights into an important biological process. [ABSTRACT FROM AUTHOR]

During development of biological organisms, multiple complex structures are formed. In many instances, these structures need to exhibit a high degree of order to be functional, although many of their constituents are intrinsically stochastic. Hence, it has been suggested that biological robustness ultimately must rely on complex gene regulatory networks and clean-up mechanisms. Here we explore developmental processes that have evolved inherent robustness against stochasticity. In the context of the Drosophila eye disc, multiple optical units, ommatidia, develop into crystal-like patterns. During the larva-to-pupa stage of metamorphosis, the centers of the ommatidia are specified initially through the diffusion of morphogens, followed by the specification of R8 cells. Establishing the R8 cell is crucial in setting up the geometric, and functional, relationships of cells within an ommatidium and among neighboring ommatidia. Here we study an PDE mathematical model of these spatio-temporal processes in the presence of parametric stochasticity, defining and applying measures that quantify order within the resulting spatial patterns. We observe a universal sigmoidal response to increasing transcriptional noise. Ordered patterns persist up to a threshold noise level in the model parameters. In accordance with prior qualitative observations, as the noise is further increased past a threshold point of no return, these ordered patterns rapidly become disordered. Such robustness in development allows for the accumulation of genetic variation without any observable changes in phenotype. We argue that the observed sigmoidal dependence introduces robustness allowing for sizable amounts of genetic variation and transcriptional noise to be tolerated in natural populations without resulting in phenotype variation. [ABSTRACT FROM AUTHOR]

Recent advances in stem-cell technologies include the differentiation of human embryonic stem cells (hESCs) into organ-like structures (organoids). These organoids exhibit remarkable self-organization that resembles key aspects of in vivo organ development. However, organoids have an unpredictable anatomy, and poorly reflect the topography of the dorsoventral, mediolateral, and anteroposterior axes. In vivo the temporal and the spatial patterning of the developing tissue is orchestrated by signaling molecules called morphogens. Here, we used morphogen-soaked beads to influence the spatial identities within hESC-derived brain organoids. The morphogen- and synthetic molecules-soaked beads were interpreted as local organizers, and key transcription factor expression levels within the organoids were affected as a function of the distance from the bead. We used an on-chip imaging device that we have developed, that allows live imaging of the developing hESC-derived organoids. This platform enabled studying the effect of changes in WNT/BMP gradients on the expression of key landmark genes in the on-chip human brain organoids. Titration of CHIR99201 (WNT agonist) and BMP4 directed the expression of telencephalon and medial pallium genes; dorsal and ventral midbrain markers; and isthmus-related genes. Overall, our protocol provides an opportunity to study phenotypes of altered regional specification and defected connectivity, which are found in neurodevelopmental diseases.

The blastocyst inner cell mass (ICM) that gives rise to a whole embryo in vivo can be derived and cultured in vitro as embryonic stem cells (ESCs), which retain full developmental potential. ICM cells receive, from diverse sources, complex molecular and spatiotemporal signals that orchestrate the finely-tuned processes associated with embryogenesis. Those instructions come, continuously, from themselves and from surrounding cells, such as those present in the trophectoderm and primitive endoderm (PrE). A key component of the ICM niche are the extracellular vesicles (EVs), produced by distinct cell types, that carry and transfer key molecules that regulate target cells and modulate cell renewal or cell fate. A growing number of studies have demonstrated the extracellular circulation of morphogens, a group of classical regulators of embryo development, are carried by EVs. miRNAs are also an important cargo of the EVs that have been implicated in tissue morphogenesis and have gained special attention due to their ability to regulate protein expression through post-transcriptional modulation, thereby influencing cell phenotype. This review explores the emerging evidence supporting the role of EVs as an additional mode of intercellular communication in early embryonic and ESCs differentiation.

How does pattern formation occur accurately when confronted with tissue growth and stochastic fluctuations (noise) in gene expression? Dorso-ventral (D-V) patterning of the mandibular arch specifies upper versus lower jaw skeletal elements through a combination of Bone morphogenetic protein (Bmp), Endothelin-1 (Edn1), and Notch signaling, and this system is highly robust. We combine NanoString experiments of early D-V gene expression with live imaging of arch development in zebrafish to construct a computational model of the D-V mandibular patterning network. The model recapitulates published genetic perturbations in arch development. Patterning is most sensitive to changes in Bmp signaling, and the temporal order of gene expression modulates the response of the patterning network to noise. Thus, our integrated systems biology approach reveals non-intuitive features of the complex signaling system crucial for craniofacial development, including novel insights into roles of gene expression timing and stochasticity in signaling and gene regulation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Here we discuss the formation of phyllotactic patterns in the shoot apical meristem (SAM) of plants, where the spatial distribution of the phytohormone auxin determines phyllotaxis in a domain that is growing and changing in time. We assume that the concentration of auxin modifies the mechanical properties of the domain and that the mechanical stress field in the SAM orients the flux of auxin. To study this problem we propose a mechanism for pattern formation in growing domains with variable curvature. The dynamics of chemicals is modeled by a reaction-diffusion system that produces a three dimensional pattern of chemical concentrations that changes the stress field in the domain while growing. The growth process is modeled by a phase-field order parameter which determines the location of the boundaries of the domain. This field is coupled to the chemical concentration through a curvature term that affects the local mechanical stress in the domain. The local stress changes in turn modify the chemical patterns. Our model constitutes a useful and novel approach in theoretical biology, as many developmental processes in organisms seem to be affected by the changes of curvature, size, mechanical stress and other physical aspects. Several patterns seen in many plants are reproduced under certain conditions by our model. [ABSTRACT FROM AUTHOR]

Chemical and mechanical pattern formation is fundamental during embryogenesis and tissue development. Yet, the underlying molecular and cellular mechanisms are still elusive in many cases. Most current theories assume that tissue development is driven by chemical processes: either as a sequence of chemical patterns each depending on the previous one, or by patterns spontaneously arising from specific chemical interactions (such as “Turing-patterns”). Within both theories, mechanical patterns are usually regarded as passive by-products of chemical pre-patters. However, several experiments question these theories, and an increasing number of studies shows that tissue mechanics can actively influence chemical patterns during development. In this study, we thus focus on the interplay between chemical and mechanical processes during tissue development. On one hand, based on recent experimental data, we develop new mechanochemical simulation models of evolving tissues, in which the full 3D representation of the tissue appears to be critical for obtaining a realistic mechanochemical behaviour. The presented modelling approach is flexible and numerically studied using state of the art finite element methods. Thus, it may serve as a basis to combine simulations with new experimental methods in tissue development. On the other hand, we apply the developed approach and demonstrate that even simple interactions between tissue mechanics and chemistry spontaneously lead to robust and complex mechanochemical patterns. Especially, we demonstrate that the main contradictions arising in the framework of purely chemical theories are naturally and automatically resolved using the mechanochemical patterning theory. [ABSTRACT FROM AUTHOR]