The robust patterning of cell fates during embryonic development requires the precise coordination of signalling gradients within defined spatial constraints. Using a geometrically confined in vitro system derived from human embryonic stem cells, we demonstrate that patterning of neuromesodermal progenitors during axial elongation is driven by boundary-dependent mechanisms. Despite extensive work on radial fate patterning in confined 2D systems, the quantitative role of boundary conditions in shaping spatiotemporal dynamics remains unclear. Here, we show that a minimal reaction-diffusion model coupled with a simplified gene regulatory network accurately predicts spatial patterns across diverse geometries. Guided by its predictions, we identify Wnt signalling as a key component of the activator signal. Inhibition of Wnt secretion preserved initiation of patterning but disrupted subsequent morphogenesis, indicating that distinct mechanisms govern pattern establishment versus maintenance. Our findings reveal how geometry encodes positional information that directs molecular patterning, providing insight into how spatial constraints and signalling dynamics guide robust tissue self-organisation during development.

Cells take on specific fates during development based on cues, which can be genetic or mechanical. Now it is shown that the decision of cell nuclei to either migrate to the outer cortex or remain internalized in fruit fly embryos depends on topology.

During development and tissue repair, cells reshape and reconfigure to ensure organs take specific shapes. This process is inherently three-dimensional (3D). Yet, in part due to limitations in imaging and data analysis, cell shape analysis within tissues has largely been studied in two-dimensions (2D), e.g., the Drosophila wing disc. With recent advances in imaging and machine learning, there has been significant progress in our understanding of 3D cell and tissue shape in vivo. However, even after gaining 3D segmentation of cells, it remains challenging to extract cell shape metrics beyond volume and surface area for cells within densely packed tissues. To address the challenge of extracting 3D cell shape metrics from dense tissues, we have developed the Python package CellMet. This user-friendly tool enables extraction of quantitative shape information from 3D cell and tissue segmentations, including cell face properties, cell twist, and cell rearrangements in 3D. Our method will improve the analysis of 3D cell shape and the understanding of cell organisation within tissues. Our tool is open source, available at https://github.com/TimSaundersLab/CellMet.

A midline in the developing central nervous system allows symmetric distribution of neural progenitors that later establish functional, bilaterally symmetric neural circuits. In the zebrafish hindbrain, a midline forms early during neurulation as a result of coordinated cell convergence and midline-crossing cell divisions (C-divisions). These processes are controlled by the Wnt/planar cell polarity (PCP) pathway that positions progenitors close to a presumptive midline to perform C-divisions. Other upstream cues that control the extent of neural plate convergence, however, remain unclear. Midkine (Mdk) and pleiotrophin (Ptn) are structurally related heparin-binding growth factors that are dynamically expressed in the developing hindbrain. We show that two zebrafish Mdks, Mdka and Mdkb, as well as Ptn interact with distinct affinities in vivo with the protein tyrosine phosphatase receptor Ptprz1b. Zebrafish mdka and ptprz1b mutants exhibit impaired neural plate convergence along with misplaced C-divisions, defective cell polarity and transiently duplicated midlines. These defects are absent in mdka; mdkb double mutants suggesting antagonistic roles of Mdka and Mdkb to coordinate convergence and C-divisions. Overexpression of Drosophila Prickle, a key component of the Wnt/PCP pathway, rescued the midline duplications in mdka and ptprz1b mutants that exhibited significantly reduced levels of prickle pk1b, pk2a, and pk2b expression. Ptprz1b overexpression, on the other hand, up-regulated pk2a transcription. Our findings therefore suggest roles for Mdka, Mdkb and Ptprz1b in coordinating neural plate convergence, neural progenitor positioning and midline formation by controlling the levels of prickle expression.

During development and wound healing, cells need to form long-range ordered structures to ensure precise formation of organs and repair damage. This requires cells to locate specific partner cells to which to adhere. How such cell matching reliably happens is an open problem, particularly in the presence of biological variability. Here, we use an equilibrium energy model to simulate how cell matching can occur with subcellular precision. A single parameter-encapsulating the competition between selective cell adhesion and cell compressibility-can reproduce experimental observations of cell alignment in the Drosophila embryonic heart. This demonstrates that adhesive differences between cells (in the case of the heart, mediated by filopodia interactions) are sufficient to drive cell matching without requiring cell rearrangements. The biophysical model can explain observed matching defects in mutant conditions and when there is significant biological variability. Using a dynamic vertex model, we demonstrate the existence of an optimal range of effective cell rigidities for efficient matching. Overall, this work shows that equilibrium energy considerations are consistent with observed cell matching in cardioblasts and has potential application to other systems, such as neuron connections and wound repair.

The transcription factor Bicoid (Bcd) establishes patterning in the early Drosophila blastocyst through its graded concentration along the anterior-posterior (AP) axis. bcd mRNA is maternally deposited during oogenesis, yet precisely when and where bcd mRNA is translated remains an open problem. Here, we take advantage of the SunTag reporter system to quantitatively examine the spatiotemporal profile of bcd mRNA translation in vivo. We demonstrate that Processing body (P body) localisation in the oocyte suppresses premature bcd mRNA translation. Upon egg laying, bcd mRNA disassociates from P bodies and translation is observed exclusively in the anterior pole of the embryo. Accompanying nuclear migration to the embryo cortex at nuclear cycle (n.c.) 9, bcd mRNA relocates to the apical domain of the nuclear environment and translation continues near the embryo surface. In n.c. 14, bcd mRNA localises to newly formed P bodies and further translation is not detected. We use these observations to build a modified source-diffusion-degradation model of Bcd gradient formation that leads to an exponential gradient by n.c. 12 and is consistent with other experimental measurements of Bcd dynamics. Consequently, we see that the spatiotemporal dynamics of bcd mRNA translation are highly regulated throughout oogenesis and early embryo development. ### Competing Interest Statement The authors have declared no competing interest.

Microtubule asters are essential in localizing the action of microtubules in processes including mitosis and organelle positioning. In large cells, such as the one-cell sea urchin embryo, aster dynamics are dominated by hydrodynamic pulling forces. However, in systems with more densely positioned nuclei such as the early Drosophila embryo, which packs around 6000 nuclei within the syncytium in a crystalline-like order, it is unclear what processes dominate aster dynamics. Here, we take advantage of a cell cycle regulation Drosophila mutant to generate embryos with multiple asters, independent from nuclei. We use an ex vivo assay to further simplify this biological system to explore the forces generated by and between asters. Through live imaging, drug and optical perturbations, and theoretical modeling, we demonstrate that these asters likely generate an effective pushing force over short distances.

Myocyte fusion is a pivotal process in the development and regeneration of skeletal muscle. Failure during fusion can lead to a range of developmental as well as pathological consequences. This review aims to comprehensively explore the intricate processes underlying myocyte fusion, from the molecular to tissue scale. We shed light on key players, such as the muscle-specific fusogens - Myomaker and Myomixer, in addition to some lesser studied molecules contributing to myocyte fusion. Conserved across vertebrates, Myomaker and Myomixer play a crucial role in driving the merger of plasma membranes of fusing myocytes, ensuring the formation of functional muscle syncytia. Our multiscale approach also delves into broader cell and tissue dynamics that orchestrate the timing and positioning of fusion events. In addition, we explore the relevance of muscle fusogens to human health and disease. Mutations in fusogen genes have been linked to congenital myopathies, providing unique insights into the molecular basis of muscle diseases. We conclude with a discussion on potential therapeutic avenues that may emerge from manipulating the myocyte fusion process to remediate skeletal muscle disorders.

Morphogens are intercellular signaling molecules providing spatial information to cells in developing tissues to coordinate cell fate decisions. The spatial information is encoded within long-ranged concentration gradients of the morphogen. Direct measurement of morphogen dynamics in a range of systems suggests that local and global diffusion coefficients can differ by orders of magnitude. Further, local diffusivity can be large, which would potentially abolish any concentration gradient rapidly. Such observations have led to alternative transport models being proposed, including transcytosis and cytonemes. Here, we show that accounting for tissue architecture combined with receptor binding is sufficient to hinder the diffusive dynamics of morphogens, leading to an order of magnitude decrease in the effective diffusion coefficient from local to global scales. In particular, we built a realistic in silico architecture of the extracellular spaces of the zebrafish brain using light and electron microscopy data. Simulations on realistic architectures demonstrate that tortuosity and receptor binding within these spaces are sufficient to reproduce experimentally measured morphogen dynamics. Importantly, this work demonstrates that hindered diffusion is a viable mechanism for gradient formation, without requiring additional regulatory control.

During development and tissue repair, cells reshape and reconfigure to ensure organs take specific shapes. This process is inherently three-dimensional (3D). Yet, in part due to limitations in imaging and data analysis, cell shape analysis within tissues have been studied as a two-dimensional (2D) approximation, e.g., the Drosophila wing disc. With recent advances in imaging and machine learning, there has been significant progress in our understanding of 3D cell and tissue shape in vivo. However, even after gaining 3D segmentation of cells, it remains challenging to extract cell shape metrics beyond volume and surface area for cells within densely packed tissues. In order to extract 3D shape metrics, we have developed CellMet. This user-friendly tool enables extraction of quantitative shape information from 3D cell and tissue segmentation. It is developed for extracting cell scale information from densely packed tissues, such as cell face properties, cell twist, and cell rearrangements. Our method will improve the analysis of 3D cell shape and the understanding of cell organisation within tissues. Our tool is open source, available at https://github.com/TimSaundersLab/CellMet. ### Competing Interest Statement The authors have declared no competing interest.

Morphogens are intercellular signaling molecules providing spatial information to cells in developing tissues to coordinate cell fate decisions. The spatial information is encoded within long-ranged concentration gradients of the morphogen. Direct measurement of morphogen dynamics in a range of systems suggests that local and global diffusion coefficients can differ by orders of magnitude. Further, local diffusivity can be large, which would potentially abolish any concentration gradient rapidly. Such observations have led to alternative transport models being proposed, including transcytosis and cytonemes. Here, we show that accounting for tissue architecture combined with receptor binding is sufficient to hinder the diffusive dynamics of morphogens, leading to an order of magnitude decrease in the effective diffusion coefficient from local to global scales. In particular, we built a realistic in silico architecture of the extracellular spaces of the zebrafish brain using light and electron microscopy data. Simulations on realistic architectures demonstrate that tortuosity and receptor binding within these spaces are sufficient to reproduce experimentally measured morphogen dynamics. Importantly, this work demonstrates that hindered diffusion is a viable mechanism for gradient formation, without requiring additional regulatory control.SIGNIFICANCE Measurements of morphogen diffusivity vary significantly depending on experimental approach. Such differences have been used to argue against diffusion as a viable mechanism of morphogen gradient formation. Here, we demonstrate that accounting for the local tissue architecture in concert with including receptor binding is sufficient to explain a range of biological observations. This demonstrates that (hindered) diffusion-driven transport is a viable mechanism of gradient formation.### Competing Interest StatementThe authors have declared no competing interest.

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.

On a curved surface, epithelial cells can adapt to geometric constraints by tilting and by exchanging their neighbors from apical to basal sides, known as an apico-basal topological transition 1 (AB-T1). The relationship between cell tilt, AB-T1s, and tissue curvature still lacks a unified understanding. Here, we propose a general framework for cell packing in curved environments and explain the formation of AB-T1s from the perspective of strain anisotropy. We find that steep curvature gradients can lead to cell tilting and induce AB-T1s. Alternatively, pressure differences across the epithelial tissue can drive AB-T1s in regions of large curvature anisotropy. The two mechanisms compete to determine the impact of tissue geometry and mechanics on optimized cell rearrangements in three dimensions.

Background Tissues need to regenerate to restore function after injury. Yet, this regenerative capacity varies significantly between organs and between species. For example, in the heart, some species retain full regenerative capacity throughout their lifespan but human cardiac cells display a limited ability to repair the injury. After a myocardial infarction, the function of cardiomyocytes is impaired and reduces the ability of the heart to pump, causing heart failure. Therefore, there is a need to restore the function of an injured heart post myocardial infarction. We investigate in cell culture the role of the Yes-associated protein (YAP), a transcriptional co-regulator with a pivotal role in growth, in driving repair after injury.Methods We express optogenetic YAP (optoYAP) in three different cell lines. We characterised the behaviour and function of optoYAP using fluorescence imaging and quantitative real-time PCR of downstream YAP target genes. Mutant constructs were generated using site-directed mutagenesis. Nuclear localised optoYAP was functionally tested using wound healing assay.Results Utilising optoYAP, which enables precise control of pathway activation, we show that YAP induces the expression of downstream genes involved in proliferation and migration. optoYAP can increase the speed of wound healing in H9c2 cardiomyoblasts. Interestingly, this is not driven by an increase in proliferation, but by collective cell migration. We subsequently dissect specific phosphorylation sites in YAP to identify the molecular driver of accelerated healing.Conclusions This study shows that optogenetic YAP is functional in H9c2 cardiomyoblasts and its controlled activation can potentially enhance wound healing in a range of conditions.

ABSTRACT A midline in the developing central nervous system (CNS) is essential for the symmetric distribution of neural progenitors that later establish functional, bilaterally symmetric neural circuits. In the zebrafish hindbrain, a midline forms early during neurulation and requires a coordinated interplay of cell convergence and midline-crossing cell divisions (C-divisions). These two processes are controlled by the Wnt/planar cell polarity (PCP) pathway. However, upstream cues that control the timely production of PCP components remain unknown. Midkine (Mdk) and pleiotrophin (Ptn) are structurally related heparin-binding growth factors that are dynamically expressed in the developing zebrafish hindbrain. We used proximity ligation assays (PLAs) and fluorescence cross correlation spectroscopy (FCCS) in vivo to show that two zebrafish Mdks, Mdka and Mdkb, as well as Ptn interact with protein tyrosine phosphatase receptors type Z1, Ptprz1a and Ptprz1b, with distinct affinities. Ligand binding triggered Ptprz1b internalization and thereby determined the availability of signaling receptor on cell membranes. In zebrafish mdka, ptn and ptprz1b mutants, cell migration and convergence were significantly impaired during hindbrain neurulation. Impaired convergence led to misplaced C-divisions, defective cell polarity and consequently duplicated midlines. These duplications were rescued by overexpression of Drosophila Prickle, a key component of the Wnt/PCP pathway. Here, we provide evidence that zygotic Mdka controls the distribution of maternally provided Ptprz1b, which in turn is needed for transcription of zebrafish prickle1b . Our findings thus reveal a role for Mdka and Ptprz1b upstream of Wnt/PCP to coordinate neural plate convergence, neural progenitor positioning and midline formation.

SUMMARYCentral Nervous System (CNS) organogenesis is a complex process that obeys precise architectural rules. The impact that nervous system architecture may have on its functionality remains, however, relatively unexplored. To clarify this problem, we analyzed the development of the Drosophila embryonic Ventral Nerve Cord (VNC). VNC morphogenesis requires the tight control of Jun kinase (JNK) signaling, exerted in part via a negative feedback loop mediated by the dual specificity phosphatase Puckered, in a subset of pioneer neurons. Here we show that the JNK pathway autonomously regulates neuronal electrophysiological properties without affecting synaptic vesicle transport. Manipulating, during early embryogenesis, JNK signaling activity in pioneer neurons, directly influences their function as ‘organizers’ of VNC architecture and, moreover, uncovers a role in the coordination of the embryonic motor circuitry that is required for hatching. Together, our data reveal critical links, mediated by the control of the JNK signaling cascade by Puckered, between the structural organization of the VNC and its functional optimization.

Morphogenesis of the Central Nervous System (CNS) is a complex process that obeys precise architectural rules. Yet, the mechanisms dictating these rules remain unknown. Analyzing morphogenesis of the Drosophila embryo Ventral Nerve Cord (VNC), we observe that a tight control of JNK signaling is essential for attaining the final VNC architecture. JNK signaling in a specific subset of pioneer neurons autonomously regulates the expression of Fasciclin 2 (Fas 2) and Neurexin IV (Nrx IV) adhesion molecules, probably via the transcription factor zfh1 . Interfering at any step in this cascade affects fasciculation along pioneer axons, leading to secondary cumulative scaffolding defects during the structural organization of the axonal network. The global disorder of architectural landmarks ultimately influences nervous system condensation. In summary, our data point to JNK signaling in a subset of pioneer neurons as a key element underpinning VNC architecture, revealing critical milestones on the mechanism of control of its structural organization.

Central nervous system organogenesis is a complex process that obeys precise architectural rules. The impact that nervous system architecture may have on its functionality remains, however, relatively unexplored. To clarify this problem, we analyze the development of the Drosophila embryonic Ventral Nerve Cord (VNC). VNC morphogenesis requires the tight control of Jun kinase (JNK) signaling in a subset of pioneer neurons, exerted in part via a negative feedback loop mediated by the dual specificity phosphatase Puckered. Here we show that the JNK pathway autonomously regulates neuronal electrophysiological properties without affecting synaptic vesicle transport. Manipulating JNK signaling activity in pioneer neurons during early embryogenesis directly influences their function as organizers of VNC architecture and, moreover, uncovers a role in the coordination of the embryonic motor circuitry that is required for hatching. Together, our data reveal critical links, mediated by the control of the JNK signaling cascade by Puckered, between the structural organization of the VNC and its functional optimization.

Immigration of mesenchymal cells into the growing fin and limb buds drives distal outgrowth, with subsequent tensile forces between these cells essential for fin and limb morphogenesis. Morphogens derived from the apical domain of the fin, orientate limb mesenchyme cell polarity, migration, division and adhesion. The zebrafish mutant stomp displays defects in fin morphogenesis including blister formation and associated loss of orientation and adhesion of immigrating fin mesenchyme cells. Positional cloning of stomp identifies a mutation in the gene encoding the axon guidance ligand, Slit3. We provide evidence that Slit ligands derived from immigrating mesenchyme act via Robo receptors at the apical ectodermal ridge (AER) to promote release of sphingosine-1-phosphate (S1P). S1P subsequently diffuses back to the mesenchyme to promote their polarisation, orientation, positioning and adhesion to the interstitial matrix of the fin fold. We thus demonstrate the coordination of the Slit-Robo and S1P signalling pathways in fin fold morphogenesis. Our work introduces a mechanism regulating the orientation, positioning and adhesion of its constituent cells.