Developmental Cell

During organogenesis, epithelial tissues undergo extensive three-dimensional (3D) remodeling while simultaneously generating specialized cell types. Whether these transient architectural states actively instruct lineage allocation remains unclear. Here we identify a morphogenetic stage in which resolution of epithelial stratification is required for lineage allocation and establishment of endocrine cell mass. We show that loss of the Hippo pathway regulator Merlin disrupts lumen morphogenesis and prevents formation of the transient 3D epithelial architecture that characterizes normal pancreas development. Failure to establish this architectural state alters lineage allocation, impairing acinar differentiation, markedly reducing adult endocrine cell mass, and disrupting glucose homeostasis. Mosaic analyses reveal that these lineage defects arise non-cell autonomously, demonstrating that epithelial architecture itself instructs cell fate decisions. Mechanistically, Merlin coordinates PI3K-regulated polarized membrane trafficking required for apical membrane biogenesis and lumen formation. Together, these findings identify Merlin-dependent membrane trafficking as a mechanism coupling epithelial morphogenesis to lineage allocation and demonstrate that transient developmental architectures can determine the cellular composition of mature organs.

Developing embryos have a striking ability to buffer external and internal perturbations. A key example of this phenomenon across developing systems is successful organ formation even after substantial cell loss. Although molecular regulators of this robustness are beginning to be understood, it remains unclear whether and how surviving cells can remodel their developmental trajectories to safeguard morphogenesis. To address this question, we use the zebrafish retina, where progenitor behaviour and lineage transitions are highly tractable and extensively characterized. We induce widespread apoptosis via heat stress and genetic approaches and track cellular and tissue-wide responses in vivo over time. We find that retinal development is highly resilient with neurogenesis initiating on time and growth continuing despite extensive apoptosis. Continued growth is supported by neurogenic progenitors that react to apoptosis through a non-cell autonomous switch in behaviour. These cells bypass their canonical differentiation route and undergo self-renewing divisions that expand clonal output and compensate for lost cells. Importantly, self-renewal is transient, progenitors resume lineage progression, generating appropriate neuronal cell types. This adaptive response supports the formation of retinas with proper architecture, connectivity to the brain and visual function. Together, these findings identify latent plasticity in the neurogenic programme as a mechanism that contributes to developmental robustness under stress.

How a conserved embryonic organiser--the Sonic hedgehog (Shh)-expressing Zone of Polarising Activity (ZPA)--generates diverse limb architectures in amniotes remains a central problem in evolutionary and developmental biology. The principal difference across species lies in the number of digits produced from ZPA tissue: two in mammals1, one in the chick leg2 and none in the chick wing2. Here we show that this divergence is governed by a Shh-p27Kip1 pathway operating in avian, but not mammalian ZPAs. In chick wing ZPA explants, attenuation of this pathway reveals an intrinsic digit-forming programme, enabling cells to self-organise signalling networks and generate digits after grafting into a host wing bud. Guided by these findings, we redirected the chick leg, which retains stem amniote digit identities, to follow a mammalian-like developmental trajectory. Precise temporal restriction of Shh signalling transforms chick legs into pentadactyl limbs with digit identities characteristic of mammals and their therapsid ancestors, with two digits arising from the ZPA. These findings establish a unifying framework for how Shh controls both digit number and identity across amniotes.

Cell extrusion is a fundamental process in tissue homeostasis, morphogenesis, and cancer progression, facilitating the removal of cells either alive or through apoptosis. While biochemical signaling pathways are known to regulate extrusion, recent advances have underscored the importance of mechanical forces in this process. Here, using optogenetic control of RhoA activation in epithelial monolayers combined with Bayesian Inversion Stress Microscopy (BISM) and three-dimensional cell-based modeling, we uncover a counterintuitive mechanism whereby elevated tension, instead of stabilizing the monolayer, actively drives extrusion in highly contractile cells. We show that local RhoA activation enhances myosin II-dependent contractility and F-actin reorganization, which promotes cell stiffening, resulting in localized tension buildup. The ensuing tensile stress amplifies vertical mechanical fluctuations, which in turn trigger cell extrusion. Remarkably, these tension-induced extrusions occur both apically and basally. Furthermore, our findings show that RhoA-mediated contractility is not merely an effector of extrusion but also an active promoter of basal extrusion, independently of caspase activation. Our study demonstrates that tensile stress can directly initiate extrusion events and bias their outcome toward apical or basal fates. By identifying tension as a driver rather than a suppressor of extrusion, this work revises current models of epithelial homeostasis and highlights mechanical control as a targetable axis in disease and regeneration.

A central dilemma of epithelial cell turnover is eliminating and replacing cells while simultaneously preserving tissue architecture and barrier function. Conventionally, apoptotic or non-apoptotic cell extrusion has been implicated in the intestinal epithelial turnover. Here, we identify a non-lytic membrane permeabilization program that drives physiological enterocyte turnover in vivo. In the Drosophila intestine, enterocytes undergo erebosis, a non-apoptotic form of cell death characterized by depletion of cytoplasmic proteins. We discover that this process is mediated by transient plasma membrane pores with estimated diameters of 16-50 nm, permitting extracellular protein influx and loss of cytoplasmic contents. The pore-forming protein Ninjurin A (NijA) accumulates as puncta during erebosis, and is necessary and sufficient for driving this process. NijA-mediated transient permeabilization preserves the membrane framework of dying cells, enabling their replacement without disrupting epithelial barrier architecture.

Oxygen is indispensable for survival in aerobic organisms, necessitating mechanisms to sense and respond to fluctuations in oxygen availability. Physiological processes such as early development, proceeds in an oxygen-sensitive environment and this appears conserved across vertebrate evolution. Owing to their avascular nature epithelial tissues routinely experience hypoxia but the epithelial responses to hypoxia and the underlying adaptive molecular regulation remains to be fully understood. We used the bilayered epidermis of Zebrafish embryos to ask how a developing epithelium responds to and copes with hypoxia. We show that under hypoxic conditions, despite the changes in cell morphologies, disruption in E-cadherin polarisation and the presence of intercellular gaps in the outer epidermal layer, the tight junctions are maintained. Our data indicate that ROCK (Rho-associated kinase) mediates the change in cell morphology and the maintenance of barrier function via non-muscle Myosin-II (NM-II). Furthermore, a high level of NM-II activity is essential to suppress Crb3-dependent cell delamination and apoptosis under hypoxia. Genetic perturbations reveal that neither increasing levels of active NMII nor augmenting tight junctions alone improves barrier function defects, indicating both these ROCK-dependent processes are necessary to maintain the barrier function under hypoxia. Our study uncovers the hitherto unappreciated importance of ROCK signaling in the maintenance of epithelial architecture and barrier function in a developing epithelium, ensuring organism survival.

Human cardiac injury responses are governed by dynamic interacting processes that are difficult to resolve. Unlike adults, fetal mammalian hearts regenerate through coordinated remodeling and proliferation supported by a pro-regenerative immune environment, extracellular matrix (ECM), and immature cardiomyocytes, including trabecular subtypes. Here, we establish a modular human cardioid injury platform to dissect these interactions. We show that anti-inflammatory macrophages selectively migrate to the injury, clear debris, and promote ECM remodeling, whereas inflammatory macrophages suppress cardiomyocyte proliferation. Synergistic FGF2-NRG1 signaling induces trabecular identity and morphology in a hyaluronan-dependent manner, conferring enhanced injury repair, characterized by cytoskeletal remodeling and cardiomyocyte proliferation mediated by YAP and WNT signaling. Exogenous YAP, but not WNT, is sufficient to promote repair in non-trabecular cardioids. These findings uncover coordinated immune-ECM-cardiomyocyte interactions governing human fetal regenerative competence and mechanistically resolve remodeling and proliferative components of cardiac repair. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/735236v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@7ff079org.highwire.dtl.DTLVardef@184d5bdorg.highwire.dtl.DTLVardef@1ec775borg.highwire.dtl.DTLVardef@190008e_HPS_FORMAT_FIGEXP M_FIG C_FIG

During suspended animation, organisms must preserve cellular organization despite the collapse of the active biochemical processes that normally maintain it. Here we show that anoxia-induced suspended animation drives cell polarity proteins into a poised, memory-like state that preserves spatial information and templates rapid resumption of morphogenesis upon reanimation. In C. elegans embryos, anoxia drives progressive assembly of the polarity protein PAR-3 into solid-like clusters that preserve the polarity axis during metabolic arrest despite inactivation of patterning reactions normally required to maintain PAR asymmetry. Embryos expressing cluster-defective PAR-3 fail to maintain asymmetry during arrest and consequently exhibit polarity axis defects upon reanimation, demonstrating that arrested PAR-3 clusters function as physical templates for re-establishment of polarity. Our data suggest that cells cope with transient metabolic arrest by reversibly converting actively maintained biochemical patterns into stable physical templates that preserve spatial information for later reactivation.

The intestinal epithelium is continuously subjected to a variety of mechanical forces, including extrinsic peristaltic contractions and intrinsic tensile forces generated by epithelial cell migration. Yet, how these mechanical cues influence the cellular processes underlying intestinal homeostasis remains poorly understood. In this study, we examine the impact of mechanical forces on intestinal cell dynamics by applying controlled external stretch to intestinal organoids, combined with high-throughput single-cell transcriptomic profiling. Our analyses reveal that prolonged cyclic mechanical strain alters the composition of differentiated intestinal cell populations. Specifically, we identify a strain-induced shift in the absorptive lineage towards a less mature state, with expansion of the population of early-stage enterocytes at the crypt-villus interface. This shift is associated with downregulation of transcriptional programs controlling enterocyte maturation within absorptive precursor populations. Our findings indicate that mechanical strain directs the maturation of the intestinal absorptive lineage, and highlight a role for mechanical forces in shaping intestinal epithelial composition and function.

Neuronal migration is essential for establishing functional tissue architecture in the developing central nervous system. While cell-intrinsic mechanisms driving neuronal movement have been identified, how migratory strategies adapt to dynamic developmental tissue changes remains less understood. Here, we address this question using retinal bipolar cells generated across unlaminated and laminated stages. This enables direct comparison of neuronal translocation across tissue states. Combining quantitative live imaging with targeted perturbations, we show that the migration mode of bipolar cells switches depending on tissue lamination state. Bipolar cells born before photoreceptor layer formation undergo directed, microtubule-dependent somal translocation. In contrast, later-born cells exhibit passive, non-directed displacement driven by collective tissue movements. Interference with tissue-wide movements impairs this displacement, while disrupting photoreceptor layer formation restores directed translocation. Independent of strategy, cells achieve accurate laminar positioning, indicating that tissue context determines neuronal migration mode, a principle likely relevant across the developing neural and other tissues.

How embryos of different sizes generate reproducible body plans remains a central question in developmental biology. Do larger embryos contain more cells, or preserve conserved organizational principles that ensure robust tissue patterning independent of scale? Here, we address this question through whole-embryo quantitative mapping of cell number, tissue allocation, and spatial organization during early development in Xenopus. Using optimized in-toto 3D imaging, tissue clearing, and deep-learning for nuclei segmentation, we quantified cell numbers and reconstructed the spatial distribution of cells in early embryonic stages. Although X. laevis embryos exhibited substantially larger embryo volumes and higher total cell numbers than X. tropicalis, the proportional allocation of cells among ectoderm, mesoderm, and endoderm remained highly conserved between species. In addition, quantitative analysis of local cellular neighborhoods revealed striking conservation of spatial order, packing geometry, and large-scale tissue architecture despite major differences in embryo size and cellular density. Together, these findings demonstrate that early vertebrate embryos follow shared quantitative design principles in which embryonic scaling occurs without disruption of the underlying cellular blueprint of the body plan. Our study establishes a quantitative framework for comparing embryonic architecture across species and provides evidence that developmental organization is governed by conserved scale-invariant topological principles.

How cells sense position to adopt appropriate fates is a central problem in development. In a classic paradigm, cells read morphogen gradients encoding positional information (PI), with prolonged signal integration improving fate precision. However, how cells adapt this strategy in morphogenetic tissues remains unclear. Here, we reconstructed complete positional and signalling histories for individual cells during zebrafish neurulation, where the Sonic hedgehog (Shh) gradient patterns ventral progenitors as the neural plate folds into a tube. Despite steadily increasing Shh activity, Shh-encoded PI peaked early and then declined. Morphogenesis set this early readout window and imposed a [~]1.2-bit ceiling on Shh-encoded information about final position and fate. Fate mapping, transcriptomic analyses, and timed Shh inhibition showed that fate specification is temporally and functionally aligned with this early readout window. Thus, when morphogenesis decouples signal quality from signal strength, cells specify fate when signalling is most informative, not when signalling is strongest.

The Wnt signaling pathway is a conserved regulator of tissue patterning and regeneration, yet how cells interpret dynamic Wnt inputs to generate robust developmental outcomes remains poorly understood. Here, we establish the first optogenetically activatable Hydra line, enabling precise temporal control of canonical Wnt signaling in vivo. Optogenetic stimulation induced dose-dependent patterning phenotypes whose rate of progression scaled with stimulation intensity. Transcriptomic analysis revealed that distinct combinations of signal intensity and duration converged onto shared transcriptional trajectories and could be described by an effective exposure metric that represents the cumulative signaling input. Functionally, Wnt activation rescued head regeneration under conditions that normally prevent organizer formation, and equivalent regenerative outcomes could be achieved through either strong, short-lived stimulation or weaker, prolonged activation. Together, our results indicate that Hydra tissues decode Wnt signaling through temporal integration of cumulative pathway activity, progressively accumulating transcriptional responses until patterning thresholds are reached. These findings establish a quantitative framework for understanding how dynamic morphogen signaling is translated into stable developmental decisions during regeneration.

While optic cup folding is known to involve specific geometrical changes of RPE cells, the precise gene regulatory mechanisms orchestrating the adoption of their highly rigid geometry, and how these contribute to successful folding, remain poorly understood. To address this gap, we investigated how the increase in mechanical tension and maintenance of an elongated geometry depend on the activation of the Wnt/{beta}-catenin and YAP pathways in RPE cells. We demonstrated that interference with these pathways causes folding failure due to a reduction in RPE cellular tension. We also identified transcriptional programs controlled by these pathways that regulate the mechanical properties of the actin cytoskeleton, cell-to-cell and cell-to-ECM adhesions, and endocytosis. Finally, we hypothesized that the LINC complex, which transmits tension between the cell and nuclear membranes, is responsible for the nuclear entry of {beta}-catenin and YAP in a cellular geometry-dependent manner. We combined quantitative imaging, functional analysis, mechanical perturbation assays, and transcriptomic analysis to generate a comprehensive view of how the coordination of mechanosensitive gene expression and changes in cellular geometries drive eye formation.

In roots, the endodermis controls nutrient entry by forming a barrier known as the Casparian strip1-3. Yet how barrier-associated processes interface with systemic signaling remains unclear. Here, we show that the receptor kinase SCHENGEN3 links local barrier surveillance to systemic nutrient signaling, with outputs that depend on the effective state of ligand perception. Unlike in Arabidopsis thaliana, SCHENGEN3 activation in Lotus japonicus requires a distinct cellular competence state and cannot be triggered by exogenous ligands alone, revealing evolutionary divergence in pathway deployment. Cross-species complementation uncouples systemic nitrogen signaling from Casparian strip formation, while transcriptomic and phosphoproteomic analyses reveal largely non-overlapping signaling- and barrier-associated programs that differ between agar and agricultural soil conditions. Mechanistically, receptor-ligand comparisons, engineered receptor variants, and co-receptor mutant analyses show that systemic nitrogen signaling is retained in receptor-perception states that are insufficient to support full Casparian strip establishment. Together, these findings reveal how a shared receptor module can separate developmental and physiological outputs by linking receptor perception state to output specificity.

Necroptosis is best known as a lytic, proinflammatory cell-death pathway mediated by RIPK3 and MLKL. Effective wound repair requires the rapid resolution of inflammation, and ongoing necroptotic activity would only exacerbate tissue damage, delaying healing. However, damaged skin presents a trigger-rich environment for necroptotic signalling, an apparent paradox that remains unresolved. Using genetic ablation and pharmacological inhibition across multiple wound models, we show that inhibiting necroptosis accelerates wound closure, revealing that necroptotic signalling normally restrains repair. Surprisingly, we found that MLKL activation in wild-type keratinocytes induces differentiation and membrane repair rather than cell lysis. This adaptive, non-lethal mode of necroptotic signalling preserves barrier integrity but slows re-epithelialisation. Our findings redefine epidermal necroptotic signalling as a stress-responsive program that modulates keratinocyte fate in a trigger-rich environment. Temporarily dampening this pathway may enhance regeneration after barrier loss without compromising immune defence, revealing necroptosis as a tunable mechanism balancing tissue repair and inflammation.

Transverse asymmetric cell divisions (ACDs) in grass leaf epidermal development produce large basal pavement cells and small apical specialised cells. These "patterning divisions" generate the long-short epidermal cell pattern that is distinctive of grasses. Here, we show that patterning divisions require premitotic basal polarisation of BdPOLAR-LIKE1 (BdPL1) in the model grass Brachypodium distachyon. Loss of BdPL1 disrupted division-plane orientation and postmitotic cell-size asymmetry in all cell files, which resulted in epidermal patterning defects. Ectopic expression analyses demonstrated that BdPL1 polarisation was independent of cellular context and sufficient to promote supernumerary transverse divisions. Furthermore, the developmental regulators BdBREVIS RADIX-solo and BdYODA1 formed a post-division polarity domain enforcing cell fate asymmetry independently of BdPL1. We propose that the premitotic BdPL1 module enforces physical cell-division asymmetry contributing to medio-lateral patterning of cell types, whereas the postmitotic BdBRX-solo/BdYDA1 module enforces within-file cell fate asymmetry. Together, they robustly pattern the grass leaf epidermis.

Most of the visceral organs are anatomically asymmetric across the left-right axis. These asymmetries can be traced to a well-studied molecular cascade leading to left-sided gene expression, including Pitx2, in the mesoderm. Yet how these early differences in gene expression are converted into differential shaping of organs at later stages remains incompletely understood, and for many organs, such as the lung, the question has not even been explored. Meanwhile, the signaling pathways responsible for the morphogenesis of the lung have been intensively studied, but no insight has been reported regarding whether they should differ on the left and right sides. Here we identify Fgf10 as a Pitx2-sensitive signal in the mesenchyme of the developing mouse lung. Fgf10 expression increases as Pitx2 decreases, making the right lung, which lacks Pitx2 expression, grow faster than the left during the budding stage. Modulating Fgf10 dosage in the left mesenchyme is sufficient to alter lung budding asymmetry. At the cellular level, the faster growth of the right lung is established by increased levels of epithelial proliferation, without significant differences in directional migration into the mesenchyme. Conditional genetics further show that Pitx2 acts during the budding stage to establish later branching asymmetry. Thus, Pitx2 converts left-right mesenchymal identity into organ asymmetry by quantitatively tuning Fgf10-dependent epithelial growth during early organogenesis.

Efficient cancer cell invasion requires coordinated control of actomyosin contractility, extracellular matrix (ECM) engagement and remodeling, yet how these processes are integrated in complex, three-dimensional (3D) environments remains unclear. Here, we identify the tumor suppressor and RhoGAP protein DLC1 as molecular brake on multicellular streaming invasion in collagen-rich ECM. Using CRISPRoff-engineered breast cancer spheroids, DLC1 reconstitution models and patient-derived organoids embedded in collagen gels, we show that DLC1 downregulation promotes an efficient multicellular streaming phenotype. This invasion program requires matrix metalloproteinase activity, {beta}1 integrin engagement and Rho-ROCK-dependent actomyosin contractility. Mechanistically, DLC1 downregulation stabilized the rear-polarization of RhoA activity and increased {beta}1 integrin abundance, plasma membrane localization and activation. Separation-of-function mutants revealed that DLC1 restrains invasion through a dual mechanism: its RhoGAP activity limited actomyosin-driven streaming, whereas its LD-like talin-binding motif controlled {beta}1 integrin enrichment at the plasma membrane. Together, our findings provide a molecular basis for the prominent role of DLC1 as a metastasis suppressor.

Cell extrusion contributes to epithelial homeostasis, but its dysregulation can lead to tumorigenesis or degeneration. A fine balance in this process is therefore essential for tissue integrity. Yet the cell types and states vulnerable to extrusion, and the mechanisms that drive it, remain elusive. Here, using spatial maps of cell states in human idiopathic pulmonary fibrosis (IPF) we find that aberrant TP53 activation in alveolar epithelial cells drives cell extrusion. Genetic modulation of TP53 specifically in alveolar epithelial type 1 cells (AT1) was sufficient to induce plasticity and subsequent extrusion as demonstrated by lineage tracing and live imaging. Strikingly, single cell and bulk transcriptome profiling revealed aberrant TP53 drives AT1 cells to acquire a transitional state mirroring AT2-derived regeneration associated intermediate states. Critically, loss of AT1 derived transitional state triggers a compensatory AT2-derived regenerative response, establishing a bidirectional transitional state that activates myofibroblasts and remodels the alveolus. Together, our study implicates AT1 plasticity and their reversion as an unrecognized driver of epithelial cell loss and establishes bidirectional transitional state as a central mechanism underlying progression of fibrotic remodeling.