Developmental Cell

Apical cell extrusion is a mechanical process that allows epithelia to eliminate apoptotic cells and prevent inflammation. Mechanosensitive ion channels are often invoked for their capacity to mediate rapid mechanical responses in dynamic morphogenetic processes. Here we report an unexpected strategy for PIEZO1 to support apoptotic extrusion. PIEZO1 inhibition blocks extrusion in cultured cells and zebrafish larvae. However, although PIEZO1 mediates calcium signals during the extrusion process, we show that extrusion is instead antagonized by increase in the preexisting mechanical tension of the epithelium when PIEZO1 is disrupted. Correcting enhanced pre-stress in PIEZO1-disrupted epithelia is sufficient to rescue apoptotic extrusion, even though it does not restore dynamic calcium signals. PIEZO1 supports mechanical homeostasis through a calcium/calcineurin-dependent pathway that protects MYPT1/myosin phosphatase from degradation to limit Myosin II activation. Therefore, PIEZO1 support the morphogenetic process of apoptotic extrusion through mechanical homeostasis.

The mammalian eye develops in concert with coordinated growth and remodeling of three vascular networks: the hyaloid vasculature, the choroid and retinal plexus. While retinal and choroidal systems support visual function in the mature eye, the hyaloid network plays a vital yet temporary role supporting the developing lens and inner retina. Regression of the hyaloid network is essential for optical clarity, yet the mechanisms guiding the process remain incompletely understood. Using single-cell RNA sequencing, we show that postnatal mouse hyaloid cells are broadly senescent. Hyaloid vascular smooth muscle, endothelial and immune cells display cell-cycle arrest marked by Cdkn1a with the expression of SASP factors. Genetic ablation of Cdkn1a impedes normal hyaloid regression, demonstrating that developmental senescence is essential for vascular remodeling and functions alongside apoptosis and macrophage-mediated clearance. These findings identify an unrecognized senescence-driven mechanism orchestrating hyaloid involution during ocular development, broadening the understanding of vascular remodeling in the eye.

How endothelial cells from distinct developmental sources are integrated into a single continuous vascular system remains unresolved. Here, using the developing mouse lung, we identify a mesenchymal progenitor population that generates endothelial cells de novo and incorporates them into the expanding vasculature through a mechanism we term integrative vasculogenesis. Genetic lineage tracing shows that these progenitors contribute directly to the pulmonary endothelium, defining a source distinct from endothelial cells of the major vessels. Live imaging and single-cell tracking reveal that newly specified angioblasts exhibit high motility, dispersing through stochastic migration before integrating into pre-existing vascular networks. Cell ablation demonstrates that pre-existing networks are required to support the migration, proliferation and survival of nascent endothelial cells. Integrative vasculogenesis is thus distinct from classical vasculogenesis and angiogenesis, providing a framework for how endothelial populations of different origins are assembled into a functional circulatory system.

Vertebrate heart development requires precise regulation of morphogenetic movements and dysregulation causes congenital heart defects. However, cell polarity remodeling during early heart tube formation remains underexplored. Here, we show that zebrafish ventricular cardiomyocyte precursors (vCPCs) undergo striking polarity reorientation as they transition from a single-layered epithelium to a transient double-layered configuration by involution. During this process, vCPCs remove inner adherens components and re-establish polarity toward the ECM-attached outer surface, enabling proper directional extension to form the ventricle. We identify the junctional scaffold protein Afadin a (Afdna) as a critical regulator. Afdna localizes to the outer and lateral membrane to restrict Rap1 activity, facilitating Podocalyxin translocation and adhesion disassembly at the inner interface between vCPC double layers. afdna mutants exhibit a multilayered ventricle with impaired blood flow, phenocopying human congenital ventricular obliteration. Our findings uncover a polarity-based mechanism ensuring ventricular chamber formation and provide insight into ventricular obliteration.

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.

Mural cells are key regulators of vascular architecture, yet how their contractility and morphology jointly influence vessel diameter in vivo remains poorly understood. Here, using high-resolution live imaging and single-cell morphometric analysis in zebrafish, we show that vascular smooth muscle cells (vSMCs) and pericytes undergo a developmental reduction in cell size through the progressive retraction of actin-rich primary and secondary processes during vascular remodelling. By specifically manipulating RhoA activity to alter mural cell contractility and shape, we uncover vessel-specific roles for mural cells in diameter regulation. We find that vSMC contractility is dispensable for the initial constriction of the dorsal aorta but is required to stabilise its diameter following vasoconstriction and to maintain vascular tone. In contrast, pericyte contractility is dispensable for both the constriction and stabilisation of intersegmental vessels. In the brain vasculature, vessel diameter is governed by the balance between contractile force and the extent of vessel coverage by vSMCs. Together, our findings redefine the role of mural cell contractility in the control of vessel constraint, demonstrating that morphology and vessel coverage, rather than contractility alone, are key determinants of vascular diameter in vivo.

Article summaryTissue regeneration requires organized responses to damage, including clearance of cellular debris. Using a genetic ablation system in Drosophila wing imaginal discs, we show that most debris is cleared within two days despite the absence of immune cell recruitment, which is restricted by the basement membrane. In the absence of immune cells, debris clearance occurs through Draper-mediated efferocytosis and lysosomal processing by epithelial cells. Disruption of this pathway delays debris removal and impacts regeneration. Residual debris consists of a heterogeneous mix of cellular components, indicating non-selective clearance. Together, our findings identify epithelial cells as key non-professional phagocytes during regeneration. Regeneration is a coordinated process that restores tissue integrity following damage. Following injury, tissues initiate early responses, including epithelial remodeling and clearance of cellular debris. However, how debris clearance is coordinated with regenerative growth to ensure efficient tissue repair remains poorly understood. To address how early damage responses, particularly debris clearance, are coordinated with regeneration, we used a genetic ablation system in Drosophila wing imaginal discs to induce apoptosis in the pouch region. Targeted damage generates cellular debris that localizes to both the apical and basal sides of the epithelium. We show that most cellular debris is cleared within two days after damage, although some debris persists apical to the regenerating epithelium. Notably, immune cells are not recruited to the damaged tissue due to restricted access by an intact basement membrane. Instead, we discovered that debris clearance is mediated by efferocytosis, whereby neighboring hinge epithelial cells activate JNK signaling and engulf debris via lysosomal formation. Reduction of efferocytosis by mutation of the phagocytic receptor Draper delays debris removal and increases debris persistence. This impairment has a modest impact on regeneration, as measured by adult wing size. Finally, our data indicate that residual debris consists of a heterogeneous mixture of cellular components, suggesting no preferential targeting by the clearance machinery. Together, our results reveal a previously unappreciated role for epithelial cells as non-professional phagocytes for debris clearance during regeneration.

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.

Tissue wounds comprise both dead and damaged cells. In epithelial wounds, repair is accomplished by cells at the wound edges, which are themselves often damaged. In the Drosophila pupal notum, wound-adjacent epithelial cells with plasma membrane damage often fuse to form syncytia; when plasma membrane damage is prevented, syncytia do not form. Damaged cells share cytoplasm as soon as milliseconds after wounding, and fusion pores connecting cell membranes form minutes later. A genetic screen reveals that wound-induced fusion requires endocytosis machinery, and dynamin localization indicates that endocytosis preferentially targets plasma membrane removed during fusion. Endocytosis promotes cell fusion by specifically promoting fusion pore expansion, indicated by quantitative analysis of cytoplasmic sharing between cells over time. Without endocytosis-mediated cell fusion, wound healing is slowed. Together, our results support a model of damage-induced cell fusion in which plasma membrane damage initiates fusion pores and endocytosis expands fusion pores, resulting in cellular fusion as an integration of single cell damage with tissue repair.

Embryonic pausing, including forms of diapause, enables development to be reversibly suspended during adverse conditions, but how paused embryos preserve cell fate patterning remains unclear. Using zebrafish, we show that developmental pausing results in the temporary collapse of Wnt, BMP, FGF, and Nodal signaling gradients, despite broad preservation of cell identity at the transcriptional level. In parallel, pausing induces a dormancy-enriched gene expression program (DEEP), which includes the non-canonical poly(A) polymerase tent5ba. Tent5ba promotes survival through pausing, sustains DEEP expression, and is required for robust reestablishment of axial patterning upon developmental re-entry. Poly(A)-tail and transcriptional profiling further link tent5ba to the stabilization of mRNA targets associated with robust developmental outcomes, supporting a model in which transcript polyadenylation safeguards patterning fidelity through suspended embryogenesis.

Fibroblast transcriptomic states reflect physiological and pathological tissue contexts, yet the upstream determinants that stabilize these states remain poorly defined. Integrins mediate extracellular matrix (ECM) adhesion and biochemical signaling, but whether they encode mechanical constraints into stable transcriptomic programs is unclear. Using engineered mouse fibroblasts, bulk and single-cell transcriptomics, and controlled micromechanical confinement, we show that integrins can shape the transcriptomic landscape. The bulk transcriptome of fibroblasts expressing V- and {beta}1-class indicates a shared mechanosensitive baseline, except when these integrin classes are expressed individually. We also found integrin-specific gene clusters, including {beta}1-class integrin-dependent enrichment of Areg, Epha7, Lhpp and Igf2r, which regulate development, regeneration and disease, and altered YAP1 targeted gene expression. At single-cell resolution under confinement, {beta}1-class integrins sustain a progenitor-associated program, whereas their loss or V-class enrichment promotes a constitutively activated state linked to injury repair and wound healing. Mechanical confinement and confinement duration further reshapes these states in an integrin-identity-dependent manner. Our findings establish integrin-identity as a determinant of how fibroblasts transduce mechanotemporal inputs from the cell surface to the nucleus.

The mammalian intestinal epithelium renews itself every few days through coordinated cell movement and extrusion along the crypt-villus axis, yet how these processes are physically organized across intact tissue remains unknown. Using long-term live imaging of intact intestinal tissue, nuclear strain mapping, and targeted perturbations of cell-cell adhesion, actomyosin contractility, and extracellular matrix adhesion, we find that epithelial cells experience a graded mechanical landscape encoded by villus geometry; tension is highest at the villus tip and decays toward the base. This tissue-scale tension gradient, generated by E-cadherin-mediated adhesion and actomyosin contractility, is the dominant predictor of collective cell velocity, outperforming force magnitude and cell density. Artificial re-establishment of a tension gradient is sufficient to reorient epithelial migration, whereas disruption of the gradient arrests both cell movement and extrusion. Epithelial renewal further requires a precise balance between this tension gradient and extracellular matrix-derived friction; excessive friction uncouples migration from extrusion, leading to pathological cell accumulation. Together, these findings reveal that intestinal epithelial homeostasis is organized by a geometry-encoded mechanical gradient that coordinates collective cell motion, extrusion, and tissue repair through a single tissue-scale framework.

Tissue injury immediately triggers immune defenses to prevent infection, a process that can paradoxically interfere with repair. Yet, how some organisms resolve this tension to fully regenerate remains poorly understood. Planarians, flatworms capable of regenerating any body part, offer a unique model for studying how robust immunity coexists with extensive regenerative capacity. Here, we show that the planarian immediate injury response is dominated by the robust upregulation of immune and stress-related genes, demonstrating that defense mechanisms are intrinsically wired into wound sensing. By uncoupling immune activation from tissue injury using exposure to heat-inactivated bacteria, we found that immune stimulation alone induced a transcriptional program mirroring central aspects of the early injury response. Prolonged immune activation led to progressive, host-driven tissue lysis that was fully reversible upon removal of the stimulus. Single-cell profiling identified distinct epidermal and phagocytic subpopulations as the central mediators of this "defense-first" response. Furthermore, we identified foxF-1-regulated phagocytes as critical drivers of immune resolution, as suppressing foxF-1 markedly increased vulnerability to noninfectious immune challenge. Finally, we demonstrated that sustained immune hyperactivation delayed regenerative progression by approximately 50%. Together, our findings establish the resolution of immune activity as a critical prerequisite for regeneration and define sustained immune activation as a fundamental constraint on tissue repair.

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.

The final limb structure reflects coordinated deployment of molecular programs, defined not only by which genes are expressed but when they are activated and silenced across time. Existing omics analyses obscure the temporal unfolding of these programs and conflate program identity with deployment timing by assuming temporal equivalence between conditions. We developed WATER (Weighted Windowed Assignment of Temporal Expression of RNA), a framework that reconstructs temporal gene expression trajectories independently within each condition, enabling direct comparison of temporal program architecture between wild-type and perturbed systems. Applying WATER to U11-null mouse forelimb development revealed that minor spliceosome inhibition redistributes genes across inappropriate temporal trajectories. Minor spliceosome inhibition causes splicing defects in minor intron-containing genes such as the PRC2 component Eed, leading to reduced H3K27me3 deposition and chromatin-transcription divergence. Single-cell RNA sequencing revealed persistence of progenitor states, impaired chondrogenic progression, and p53-dependent apoptotic checkpoint activation. Orthogonal WATER analysis of Eed-knockout stem cells recapitulated key features of chromatin gating failure, including temporal redistribution of skeletal development programs and progenitor state persistence, confirming that Eed loss alone is sufficient to produce temporal program redistribution independently of other splicing defects. Trp53 ablation in U11-null limbs partially rescued distal limb structures without correcting the underlying splicing defects, establishing that checkpoint activation amplifies rather than initiates the timing disruption. The limb retains much of its molecular toolkit but executes it in the wrong order, demonstrating that developmental failure arises from mistimed deployment of intact molecular programs. Thus, temporal program architecture is a fundamental organizing principle of morphogenesis.

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.

Direct lineage reprogramming holds therapeutic promise but often depends on transcription factor overexpression, resulting in unstable phenotypes. Here, we describe a novel strategy to convert fibroblasts into endothelial-like cells by activating lysosomal activity. Constitutively active MEK2 induces an endothelial gene program via sustained MAPK/ERK signaling, leading to enhanced vacuolar ATPase (V-ATPase) activity, lysosomal acidification, extracellular matrix degradation, and angiogenic behavior. V-ATPase inhibition impairs these effects, whereas pharmacologic activation with EN6 recapitulates key features of reprogramming and promotes nuclear translocation of TFEB, a master lysosomal regulator. Consistently, TFEB overexpression-particularly a phospho-deficient mutant-boosts lysosomal function and endothelial gene expression. These findings define a MAPK-V-ATPase-TFEB axis that drives endothelial reprogramming and highlight the lysosome as a central hub for cell fate transitions, offering an organelle-centric framework for regenerative medicine. HighlightsO_LISustained MEK2 activation reprograms fibroblasts into endothelial-like cells C_LIO_LIMEK2 enhances lysosomal acidification by upregulating V-ATPase subunits C_LIO_LIV-ATPase drives acidification and ECM remodeling for endothelial reprogramming C_LIO_LIV-ATPase activation promotes TFEB nuclear entry and endothelial gene expression C_LI

Apical-basal polarity in epithelial cells is controlled by a conserved set of polarity factors that define the apical, junctional and basolateral domains of the cell, but how these factors adapt to or control changes in domain sizes during cell shape changes remains unclear. Atypical protein kinase C (aPKC) is the main effector of apical identity, phosphorylating the lateral factors, Bazooka/Par-3, Lgl, Par-1 and Yurt to exclude them from the apical domain. Using analogue-sensitive aPKC in Drosophila follicle cells, we found that aPKC substrates differ over 100-fold in their sensitivity to inhibition, revealing a hierarchy of substrates, that is conserved in mammals, in which high-affinity substrates out-compete low-affinity substrates when aPKC activity is limiting. Mild aPKC inhibition prevents the phosphorylation of its lowest affinity substrate, Yurt. Yurt then accumulates apically by binding to Crumbs, where it activates apical constriction through Shroom, Cysts/Dp114RhoGEF, Rho kinase and Myosin. Yurt localises apically in cells that are stretched, either by morphogenesis or artificially, indicating that stretching reduces aPKC activity to trigger an antagonistic contraction. By contrast, yurt- cells fail to resist stretching. Thus, the aPKC/Yurt pathway functions as a homeostatic stretch response, in which apical and lateral epithelial polarity factors collaborate to mechanically regulate apical domain size.

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.