The vertebrate bauplan is primarily established via the formation of embryonic tissues in a head-to-tail progression. The mechanics of this elongation, which requires the presomitic mesoderm (PSM), remain poorly understood. Here, we find that avian PSM explants can elongate autonomously when physically confined in vitro, producing a pushing force promoting posterior elongation of the embryo. This tissue elongation is caused by volumetric expansion, which results from an increase in the extracellular fraction accompanied by graded cellular motility. We show that fibroblast growth factor (FGF) signaling promotes glycolysis-dependent production of hyaluronic acid (HA), which is required for expansion of the posterior PSM. Our findings link body axis elongation to tissue expansion through the metabolic control of extracellular matrix production downstream of FGF signaling.

Sparse mesenchymal cellular networks are ubiquitous across animals, shaping both embryonic and adult structures through dynamic interactions with epithelia. Yet, the physical principles underlying their collective behaviors remain elusive, as their stellate cells and large extracellular spaces-filled with matrix or interstitial fluid-pose significant experimental and computational challenges. Here, we demonstrate that the avian presomitic mesoderm (PSM), a canonical embryonic mesenchymal tissue, behaves as a fluid under tension, exhibiting structural organization that cannot be explained by simple Brownian-like cell motion. Through quantitative modeling, we identify contact inhibition of locomotion (CIL)-where cells actively retract and move away upon contact-as a key mechanism that enables sparse mesenchymal networks to sustain macroscopic tension while flowing like a fluid. Simple continuum equations relate observable cell-scale parameters to the emergent remodeling dynamics observed in both experiments and simulations. Together, these findings put forward an unrecognized mechanical role for CIL, extending its influence beyond collective migration, and establish the fluid-under-tension state as a distinct class of tissue behavior that describes key developing embryonic tissues and may illuminate how matrix-rich adult tissues become fluidized during tumorigenesis. ### Competing Interest Statement The authors have declared no competing interest. Academy of Medical Sciences, SBF009/1014 Wellcome Trust, 319420/Z/24/Z Taighde Éireann-Research Ireland, 24/PATH-S/12626 Leverhulme Trust, RPG-2024-147 Chan Zuckerberg Initiative DAF, 2023-329572

The transformation of a two-dimensional epithelial sheet into various three-dimensional structures is a critical process in generating the diversity of animal forms. Previous studies of epithelial folding have revealed diverse mechanisms driven by epithelium-intrinsic or -extrinsic forces. Yet little is known about the biomechanical basis of epithelial splitting, which involves extreme folding and eventually a topological transition breaking the epithelial tube. Here, we leverage tracheal-esophageal separation (TES), a critical and highly conserved morphogenetic event during tetrapod embryogenesis, as a model system for interrogating epithelial tube splitting. We identify an evolutionarily conserved, compressive force exerted by the mesenchyme surrounding the epithelium, as being necessary to drive epithelial constriction and splitting. The compressive force is mediated by localized convergent flow of mesenchymal cells towards the epithelium. Sonic hedgehog (SHH) secreted by the epithelium functions as an attractive cue for mesenchymal cells. Removal of the mesenchyme, inhibition of cell migration, or loss of SHH signaling all abrogate TES, which can be rescued by externally applied pressure. These results unveil the biomechanical basis of epithelial splitting and suggest plausible mesenchymal origins of tracheal-esophageal birth defects.

Marine picocyanobacteria Prochlorococcus and Synechococcus , the most abundant photosynthetic cells in the oceans, are generally thought to have a primarily single-celled and free-living lifestyle. However, while studying the ability of picocyanobacteria to supplement photosynthetic carbon fixation with the use of exogenous organic carbon, we found the widespread occurrence of genes for breaking down chitin, an abundant source of organic carbon that exists primarily as particles. We show that cells that encode a chitin degradation pathway display chitin degradation activity, attach to chitin particles, and show enhanced growth under low light conditions when exposed to chitosan, a partially deacetylated soluble form of chitin. Marine chitin is largely derived from arthropods, which underwent major diversifications 520 to 535 Mya, close to when marine picocyanobacteria are inferred to have appeared in the ocean. Phylogenetic analyses confirm that the chitin utilization trait was acquired at the root of marine picocyanobacteria. Together this leads us to postulate that attachment to chitin particles allowed benthic cyanobacteria to emulate their mat-based lifestyle in the water column, initiating their expansion into the open ocean, seeding the rise of modern marine ecosystems. Subsequently, transitioning to a constitutive planktonic life without chitin associations led to cellular and genomic streamlining along a major early branch within Prochlorococcus . Our work highlights how the emergence of associations between organisms from different trophic levels, and their coevolution, creates opportunities for colonizing new environments. In this view, the rise of ecological complexity and the expansion of the biosphere are deeply intertwined processes.

The vertebrate body displays a segmental organization that is most conspicuous in the periodic organization of the vertebral column and peripheral nerves. This metameric organization is first implemented when somites, which contain the precursors of skeletal muscles and vertebrae, are rhythmically generated from the presomitic mesoderm. Somites then become subdivided into anterior and posterior compartments that are essential for vertebral formation and segmental patterning of the peripheral nervous system(1-4). How this key somitic subdivision is established remains poorly understood. Here we introduce three-dimensional culture systems of human pluripotent stem cells called somitoids and segmentoids, which recapitulate the formation of somite-like structures with anteroposterior identity. We identify a key function of the segmentation clock in converting temporal rhythmicity into the spatial regularity of anterior and posterior somitic compartments. We show that an initial 'salt and pepper' expression of the segmentation gene MESP2 in the newly formed segment is transformed into compartments of anterior and posterior identity through an active cell-sorting mechanism. Our research demonstrates that the major patterning modules that are involved in somitogenesis, including the clock and wavefront, anteroposterior polarity patterning and somite epithelialization, can be dissociated and operate independently in our in vitro systems. Together, we define a framework for the symmetry-breaking process that initiates somite polarity patterning. Our work provides a platform for decoding general principles of somitogenesis and advancing knowledge of human development.

Tissue morphogenesis, homoeostasis and repair require cells to constantly monitor their three-dimensional microenvironment and adapt their behaviours in response to local biochemical and mechanical cues. Yet the mechanical parameters of the cellular microenvironment probed by cells in vivo remain unclear. Here, we report the mechanics of the cellular microenvironment that cells probe in vivo and in situ during zebrafish presomitic mesoderm differentiation. By quantifying both endogenous cell-generated strains and tissue mechanics, we show that individual cells probe the stiffness associated with deformations of the supracellular, foam-like tissue architecture. Stress relaxation leads to a perceived microenvironment stiffness that decreases over time, with cells probing the softest regime. We find that most mechanical parameters, including those probed by cells, vary along the anteroposterior axis as mesodermal progenitors differentiate. These findings expand our understanding of in vivo mechanosensation and might aid the design of advanced scaffolds for tissue engineering applications.

Tissue morphogenesis and repair, as well as organ homeostasis, require cells to constantly monitor their 3D microenvironment and adapt their behaviors in response to local biochemical and mechanical cues 1-6 . In vitro studies have shown that substrate stiffness and stress relaxation are important mechanical parameters in the control of cell proliferation and differentiation, stem cell maintenance, cell migration 7-11 , as well as tumor progression and metastasis 12,13 . Yet, the mechanical parameters of the microenvironment that cells perceive in vivo , within 3D tissues, remain unknown. In complex materials with strain- and time-dependent material properties, the perceived mechanical parameters depend both on the strain and timescales at which the material is mechanically probed 14 . Here, we quantify in vivo and in situ the mechanics of the cellular microenvironment that cells probe during vertebrate presomitic mesoderm (PSM) specification. By analyzing the magnitude and dynamics of endogenous, cell-generated strains, we show that individual cells preferentially probe the stiffness associated with deformations of the supracellular, foam-like tissue architecture. We reveal how stress relaxation leads to a perceived microenvironment stiffness that decreases over time, with cells probing the softest regime. While stress relaxation timescales are spatially uniform in the tissue, most mechanical parameters, including those probed by cells, vary along the anteroposterior axis, as mesodermal progenitors commit to different lineages. Understanding the mechanical parameters that cells probe in their native 3D environment is important for quantitative studies of mechanosensation in vivo 2-4,6,15 and can help design scaffolds for tissue engineering applications 16-18 .

The vertebrate anteroposterior axis forms through elongation of multiple tissues during embryogenesis. This process is based on tissue-autonomous mechanisms of force generation and intertissue mechanical coupling whose failure leads to severe developmental anomalies such as body truncation and spina bifida. Similar to other morphogenetic modules, anteroposterior body extension requires both the rearrangement of existing materials—such as cells and extracellular matrix—and the local addition of new materials, i.e., anisotropic growth, through cell proliferation, cell growth, and matrix deposition. Numerous signaling pathways coordinate body axis formation via regulation of cell behavior during tissue rearrangements and/or volumetric growth. From a physical perspective, morphogenesis depends on both cell-generated forces and tissue material properties. As the spatiotemporal variation of these mechanical parameters has recently been explored in the context of vertebrate body elongation, the study of this process is likely to shed light on the cross talk between signaling and mechanics during morphogenesis. 27.1 Review in Advance first posted on August 14, 2019. (Changes may still occur before final publication.) A nn u. R ev . C el l D ev . B io l. 20 19 .3 5. D ow nl oa de d fr om w w w .a nn ua lr ev ie w s. or g A cc es s pr ov id ed b y C am br id ge U ni ve rs ity o n 08 /1 6/ 19 . F or p er so na l u se o nl y. CB35CH27_Pourquie ARjats.cls August 7, 2019 16:15

The vertebrate anteroposterior axis forms through elongation of multiple tissues during embryogenesis. This process is based on tissue-autonomous mechanisms of force generation and intertissue mechanical coupling whose failure leads to severe developmental anomalies such as body truncation and spina bifida. Similar to other morphogenetic modules, anteroposterior body extension requires both the rearrangement of existing materials-such as cells and extracellular matrix-and the local addition of new materials, i.e., anisotropic growth, through cell proliferation, cell growth, and matrix deposition. Numerous signaling pathways coordinate body axis formation via regulation of cell behavior during tissue rearrangements and/or volumetric growth. From a physical perspective, morphogenesis depends on both cell-generated forces and tissue material properties. As the spatiotemporal variation of these mechanical parameters has recently been explored in the context of vertebrate body elongation, the study of this process is likely to shed light on the cross talk between signaling and mechanics during morphogenesis.

Just as in clay moulding or glass blowing, physically sculpting biological structures requires the constituent material to locally flow like a fluid while maintaining overall mechanical integrity like a solid. Disordered soft materials, such as foams, emulsions and colloidal suspensions, switch from fluid-like to solid-like behaviours at a jamming transition(1-4). Similarly, cell collectives have been shown to display glassy dynamics in 2D and 3D(5,6) and jamming in cultured epithelial monolayers(7,)(8), behaviours recently predicted theoretically(9-11) and proposed to influence asthma pathobiology(8) and tumour progression(12). However, little is known about whether these seemingly universal behaviours occur in vivo(13) and, specifically, whether they play any functional part during embryonic morphogenesis. Here, by combining direct in vivo measurements of tissue mechanics with analysis of cellular dynamics, we show that during vertebrate body axis elongation, posterior tissues undergo a jamming transition from a fluid-like behaviour at the extending end, the mesodermal progenitor zone, to a solid-like behaviour in the presomitic mesoderm. We uncover an anteroposterior, N-cadherindependent gradient in yield stress that provides increasing mechanical integrity to the presomitic mesoderm, consistent with the tissue transiting from a wetter to a dryer foam-like architecture. Our results show that cell-scale stresses fluctuate rapidly (within about 1 min), enabling cell rearrangements and effectively 'melting' the tissue at the growing end. Persistent (more than 0.5 h) stresses at supracellular scales, rather than cell-scale stresses, guide morphogenetic flows in fluid-like tissue regions. Unidirectional axis extension is sustained by the reported rigidification of the presomitic mesoderm, which mechanically supports posterior, fluid-like tissues during remodelling before their maturation. The spatiotemporal control of fluid-like and solid-like tissue states may represent a generic physical mechanism of embryonic morphogenesis.

The spatiotemporal patterning of physical stresses and mechanical properties inside living tissues ultimately guide morphogenesis, both during normal development and cancer progression. Here, we present various droplet-based techniques for measuring these physical quantities in biological systems, both in vivo and in vitro. We describe the methods for generating and functionalizing droplets, which can be placed in a tissue and imaged in 3D. We present an open source Matlab based computational tool recently made available to the community that performs automated 3D surface segmentation and curvature analysis from a volumetric image stack with typical errors in radial and curvature measurements less than 2 and 20 percent, respectively. By placing cell-sized fluorocarbon droplets within multicellular spheroids, we measure anisotropies in cell-generated stresses and quantify how these stresses change over time and space within the aggregates. Furthermore we present in situ calibrated measurements of cellular stresses inside the developing zebrafish tailbud (10-12 somite stage) obtained with ferrofluid droplets (30-50 microns in diameter). Ferrofluid droplets were also used to measure mechanical properties using magnetic actuation. In spheroids, stresses are observed to increase significantly over time while maintaining a weak spatial dependence. In vivo studies in the zebrafish tailbud, however, reveal no significant spatial dependence in the magnitude of cellular stresses, although patterning of the material properties is observed. This work highlights the potential for droplet based in situ measurements to elucidate mechanical phenomena underlying processes in developmental and cancer biology.

Type 2 diabetes (T2D) is a disease characterized by impaired insulin secretion. The Wnt signaling transcription factor Tcf7l2 is to date the T2D-associated gene with the largest effect on disease susceptibility. However, the mechanisms by which TCF7L2 variants affect insulin release from β-cells are not yet fully understood. By taking advantage of a tcf7l2 zebrafish mutant line, we first show that these animals are characterized by hyperglycemia and impaired islet development. Moreover, we demonstrate that the zebrafish tcf7l2 gene is highly expressed in the exocrine pancreas, suggesting potential bystander effects on β-cell growth, differentiation and regeneration. Finally, we describe a peculiar vascular phenotype in tcf7l2 mutant larvae, characterized by significant reduction in the average number and diameter of pancreatic islet capillaries. Overall, the zebrafish Tcf7l2 mutant, characterized by hyperglycemia, pancreatic and vascular defects, and reduced regeneration proves to be a suitable model to study the mechanism of action and the pleiotropic effects of Tcf7l2, the most relevant T2D GWAS hit in human populations.

Multicellular spheroids serve as an excellent platform to study tissue behavior and tumor growth in a controlled, three-dimensional (3D) environment. While molecular and cellular studies have long used this platform to study cell behavior in 3D, only recently have studies using multicellular spheroids shown an important role for the mechanics of the microenvironment in a wide range of cellular processes, including during tumor progression. Despite the well-established relevance of mechanical cues to cell behavior and the numerous studies on mechanics using 2D cell culture systems, the spatial and temporal variations in endogenous cellular forces within growing multicellular aggregates remain unknown. Using cell-sized oil droplets with controlled physicochemical properties as force transducers in mesenchymal cell aggregates, we show that the magnitude of cell-generated stresses varies only weakly with spatial location within the spherical aggregate, but it increases considerably over time during aggregate compaction and growth. Moreover, our results indicate that the temporal increase in cellular stresses is due to increasing cell pulling forces transmitted via integrin-mediated cell adhesion, consistent with the need for larger intercellular pulling forces to compact cell aggregates.

The evolutionary origins of the hypoxia-sensitive cells that trigger amniote respiratory reflexes – carotid body glomus cells, and ‘pulmonary neuroendocrine cells’ (PNECs) - are obscure. Homology has been proposed between glomus cells, which are neural crest-derived, and the hypoxia-sensitive ‘neuroepithelial cells’ (NECs) of fish gills, whose embryonic origin is unknown. NECs have also been likened to PNECs, which differentiate in situ within lung airway epithelia. Using genetic lineage-tracing and neural crest-deficient mutants in zebrafish, and physical fate-mapping in frog and lamprey, we find that NECs are not neural crest-derived, but endoderm-derived, like PNECs, whose endodermal origin we confirm. We discover neural crest-derived catecholaminergic cells associated with zebrafish pharyngeal arch blood vessels, and propose a new model for amniote hypoxia-sensitive cell evolution: endoderm-derived NECs were retained as PNECs, while the carotid body evolved via the aggregation of neural crest-derived catecholaminergic (chromaffin) cells already associated with blood vessels in anamniote pharyngeal arches.

The mechanical properties of the cellular microenvironment and their spatiotemporal variations are thought to play a central role in sculpting embryonic tissues, maintaining organ architecture and controlling cell behavior, including cell differentiation. However, no direct in vivo and in situ measurement of mechanical properties within developing 3D tissues and organs has yet been performed. Here we introduce a technique that employs biocompatible, magnetically responsive ferrofluid microdroplets as local mechanical actuators and allows quantitative spatiotemporal measurements of mechanical properties in vivo. Using this technique, we show that vertebrate body elongation entails spatially varying tissue mechanics along the anteroposterior axis. Specifically, we find that the zebrafish tailbud is viscoelastic (elastic below a few seconds and fluid after just 1 min) and displays decreasing stiffness and increasing fluidity toward its posterior elongating region. This method opens new avenues to study mechanobiology in vivo, both in embryogenesis and in disease processes, including cancer.

In gnathostome vertebrates, including fish, birds and mammals, peripheral nerves link nervous system, body and immediate environment by integrating efferent pathways controlling movement apparatus or organ function and afferent pathways underlying somatosensation. Several lines of evidence suggest that peripheral nerve assembly involves instructive interactions between efferent and afferent axon types, but conflicting findings challenge this view. Using genetic modeling in zebrafish, chick and mouse we uncover here a conserved hierarchy of axon type-dependent extension and selective fasciculation events that govern peripheral nerve assembly, which recapitulates the successive phylogenetic emergence of peripheral axon types and circuits in the vertebrate lineage.