Developmental Cell

Cell migration frequently requires cells to traverse tissue environments with distinct physical and biochemical properties. How migrating cells preserve a common migratory program while adapting to these heterogeneous environments remains poorly understood. Here, we show that chick primordial germ cells (PGCs) preserve a common bleb-based migratory mode throughout embryogenesis despite migrating through mechanically distinct tissues. PGCs formed membrane blebs during both intravascular crawling and migration through the dorsal mesentery. However, nuclear envelope (NE) unfolding and activation of the NE-cPLA2 pathway occurred specifically during migration through the mechanically confined dorsal mesentery, where this pathway was required for bleb formation and efficient migration. In contrast, bleb formation during vascular crawling occurred independently of the NE-cPLA2 pathway, demonstrating that distinct molecular mechanisms can generate the same migratory behavior in different tissue environments. Together, these findings suggest that diverse environmental cues converge on a conserved bleb-forming machinery. We propose a hierarchical model in which migrating cells preserve a common migratory program by flexibly switching the upstream molecular mechanisms that initiate bleb formation according to the tissue environment.

Between embryonic days 7.5 and 8.5, the mouse embryo undergoes a dramatic rearrangement of its entire anterior, a process known as ventral folding: the near-simultaneous morphogenesis of the cardiac crescent, cranial headfolds, and anterior foregut that together establish the antero-ventral body plan. While cardiac morphogenesis has been studied in detail, the cellular and mechanical basis of foregut involution remains largely undefined. Using combined light-sheet and spinning-disk live imaging spanning the full window of foregut formation, we show that involution proceeds through a stereotyped morphological programme that does not require actomyosin contractility for its initiation. Additionally, involution is preceded by a spatiotemporally restricted wave of apoptosis in embryonic visceral endoderm (emVE) cells, which extrude bidirectionally. A lineage- and stage-resolved bulk RNA-sequencing of emVE, definitive endoderm, and epiblast populations identifies a differential-adhesion and cell-cycle signature underlying this behaviour. Revisiting a visceral-endoderm-specific Bmp2 knock-out mutant, we find that BMP2 controls involution indirectly, by directing notochord positioning and thereby the geometry of the surrounding heart and headfold mechanics. Together, these findings reframe ventral folding as a single coordinated geometric process rather than a set of independent organ forming events.

During embryonic development, cellular competence to respond to differentiation signals changes dynamically. Although the mechanisms underlying competence acquisition have been extensively studied, those underlying competence loss remain unclear. In preimplantation mouse embryos, Hippo signaling shifts from regulating trophectoderm (TE) fate specification to promoting epiblast maturation. During the blastocyst stage, inner cell mass (ICM) cells lose TE competence in response to the Hippo signaling effector TEAD-YAP. Here, we show that the pioneer factor SOX2 terminates TE competence in the ICM. SOX2 binding to the TEAD-YAP-dependent TE enhancer (TEE) of the TE regulator Gata3 induces chromatin closure, suppressing TEE responsiveness to TEAD-YAP activity. This function of SOX2 requires its interaction with the corepressor TLE4 and histone deacetylase. Similar SOX2-dependent chromatin closure also occurs around other TE genes, including the TE enhancer of another TE regulator, Cdx2. Thus, SOX2 terminates TE competence in ICM cells by closing Hippo signaling-responsive enhancers.

The conserved core planar cell polarity (PCP) pathway orients cells and subcellular structures within an epithelium through asymmetric protein localization and intercellular communication. In vestibular organs and lateral-line neuromasts, mechanosensory hair cells are interspersed among support cells and form opposing hair-bundle orientations along a shared axis, enabling bidirectional sensitivity to head motion and water flow, respectively. In zebrafish neuromasts, Notch-mediated lateral inhibition gives rise to two hair-cell populations, distinguished by differential Emx2 expression, that orient their cell-intrinsic polarity machinery differently relative to a PCP-dependent tissue-wide axis. However, it remains unclear how PCP proteins are organized across hair cells and support cells to achieve both opposing hair-bundle orientations and tissue-wide alignment, and whether PCP signaling remains required after hair-bundle polarity is established. Combining quantitative spatial mapping of the core PCP protein Vangl2 with cell-type-specific and temporally controlled protein degradation, we show that hair cells and support cells make distinct yet coordinated contributions to the polarized Vangl2 organization within neuromasts and to bidirectional hair-bundle polarity. Support-cell Vangl2 facilitates tissue-wide alignment of hair bundles along the anteroposterior axis, whereas hair-cell Vangl2 is required to generate opposing hair-bundle orientations along this axis. Vangl2 degradation after hair bundles have formed disrupts their tissue-wide alignment, showing that planar polarity is actively maintained rather than fixed after establishment. Together, these findings reveal how Vangl2-dependent PCP signaling is distributed across distinct cell types within a heterogeneous epithelium to generate opposing polarity outcomes and remains necessary to preserve tissue-level planar organization.

Confined migration is essential for metastasis, yet how cells adapt their migratory and metabolic programs across stiffness-varying microenvironments remains unclear. We uncover a stiffness-dependent mechano-metabolic switch governing migration. In stiff microchannels, cells utilize the osmotic engine model (OEM), relying on NHE1 activity, front-polarization, and glycolysis. In soft microchannels, migration is OEM-independent and requires pyruvate-fueled oxidative phosphorylation (OxPHOS). This OxPHOS-driven motility depends on Arp3, {beta}1-integrin and integrin-linked kinase, which increase membrane tension in confinement that in turn triggers TRPM7-mediated calcium influx and RhoA-/myosin-II contractility. Activating and polarizing NHE1, via overexpression, hypoxia or elevated viscosity, restore OEM- and glycolysis-dependent migration in soft microchannels, bypassing the need for actin polymerization in vitro and in chick embryos. Mitochondria addition reinstates Arp3 polarization and enhances migration in NHE1-overexpressing cells, enabling engagement of both mechanisms in vitro and in zebrafish. These findings uncover a previously unrecognized mechano-metabolic link, revealing that intracellular rewiring overrides stiffness-dependent metabolic demands.

While cell identities are established early during embryogenesis, these cells remain immature until germination, and the mechanisms enforcing this developmental pause are poorly understood. Embryonic stomatal cells provide a model to study this pause as the stomatal transcription factor FAMA, normally sufficient for Guard Cell maturation in seedlings, can not drive maturation in the Arabidopsis embryo. Here we show that FAMAs ability to drive maturation depends on leaf polarity and adaxial stomatal cells can progress further in their lineage. We next find that ERECTA-family receptor signaling, which controls stomatal patterning, also suppresses embryonic stomatal maturation. In er erl1 erl2 mutants, cell pairs at the cotyledon tip acquire characteristics of maturing guard cells: cell wall reinforcement, pore-associated thickening, and expression of late lineage markers as identified by whole embryo transcriptomics. This precocious maturation however remains incomplete: many GC markers remain absent, and cells lack an open pore and mature vacuoles. Genetic analysis shows that partial maturation requires but is not limited by low levels of FAMA. Restriction of maturation to the cotyledon tip correlates with locally elevated ERECTA-family receptor abundance, while high auxin appears dispensable for this. Finally, we show that EPFL-ER signaling mediates leaf tip Guard Cell size postembryonically as well. Altogether, we identify ERECTA signaling as a local brake on embryonic stomatal cell maturation, discovering another way to push precocious stomatal cell maturation that results in a complex, partially mature cell state that provide insights into the limitations on cell embryonic cell maturation.

Homeostatic epithelia must balance stem cell maintenance, progenitor differentiation, and clearance of damaged cells while preserving barrier integrity. We investigated how integrin- ECM adhesion is regulated in the Drosophila midgut, a homeostatic epithelium with basal stem cells. The midgut expresses two beta integrins: ubiquitous {beta}Mys and endoderm-specific {beta}{nu}. ISCs and enteroblasts express only {beta}Mys, which pairs with Mew to mediate enteroblast attachment to the basement membrane. In contrast, enterocytes express both {beta}Mys and {beta}{nu}; Mew/{beta}{nu} supports ECM adhesion, while {beta}Mys pairs with Scab and localises to the basal labyrinth. Enterocytes lacking Mew or {beta}{nu} detach and are apically extruded, but this phenotype is rescued when the corresponding integrin is removed from the entire epithelium. Thus, enterocytes compete for basement membrane adhesion, with less adhesive cells being eliminated by their neighbours. In {beta}{nu} homozygotes, enteroblasts expand basally and adopt a migratory-like morphology. We propose that integrin-mediated competition for ECM adhesion is a general phenomenon that functions in the midgut to promote enterocyte extrusion, which stimulates the migration of nearby enteroblasts to maintain gut homeostasis.

The adult endometrium regenerates repeatedly, yet the cells and mechanisms that rebuild its epithelium remain poorly defined. To control the cell types available for regeneration, a genetic model to extensively ablate the uterine epithelium was combined with transplantation of lineage-labeled organoids. Ablation without organoid transplantation triggered re-epithelialization, but resulted in infertility. Transplanted endometrial epithelial organoids engrafted into the ablated uterus, reconstructed both the luminal and glandular epithelia, and restored fertility. Depleting organoids of the glandular lineage before transplantation revealed that luminal epithelial-derived cells acquire glandular identity and function after engraftment. The same luminal-to-glandular epithelial differentiation trajectory emerged during endogenous repair following targeted glandular ablation. Together, these findings establish luminal-to-glandular epithelial conversion as an intrinsic regenerative property of the adult uterine epithelium and establish an endometrial organoid transplantation platform with therapeutic potential.

Respiratory mechanics direct cell fate in the lung, but the mechanisms by which these mechanical signals are sensed and transmitted to the nucleus to control cell state remain unclear. We paired in vivo perturbations of respiratory mechanics with single-cell genomics and found that alveolar fibroblasts are highly sensitive to physical changes in their microenvironment, exhibiting persistent shifts in their transcriptional identity after injury. Surprisingly, transmission of these signals through the nuclear envelope was not essential for maintaining transcriptional or epigenetic stability during homeostasis. However, severing mechanical-nuclear signaling promoted the normalization of alveolar fibroblast identity after acute injury, resulting in improved epithelial regeneration and reduced dysplastic remodeling. These studies reveal the importance of mechanical-nuclear signaling in the regulation of alveolar cell identity and function and reveal that targeting this complex can enhance tissue regeneration.

Cell junctions establish and maintain epithelial architecture despite fluctuating environmental and developmental conditions. A central question is how cells respond to challenging conditions to preserve junctional integrity. Here, we report the discovery of a previously unrecognized quality control pathway that monitors epithelial junctions (J-QC). We used the Caenorhabditis elegans epidermis as a model to investigate the DLG-1-AJM-1 complex (DAC), a junctional domain that is critical for embryonic morphogenesis. We identify two mechanisms that sustain junctional integrity: first, localized dlg-1 mRNA ensures appropriate DLG-1 protein levels at the junction; repositioning dlg-1 RNA reduces DLG-1 levels, leading to gaps between epithelial cells. Second, transcription of DAC components responds to perturbations that disrupt the DAC. This response is sequence-independent, distinguishing it from other quality control mechanisms. It is activated by perturbations of the DAC or cytoskeleton and requires the LINC complex component ZYG-12/HOOK1-3 to transduce information about junctional integrity to the nucleus. These findings define a novel junctional QC for epithelial maintenance.

Cells generate dynamic patterns of Rho GTPase activation to direct the subsequent patterning of Rho GTPase effectors needed to remodel the cell cortex during processes ranging from cell division to cell repair. To understand how such patterns arise, we used live cell imaging, time-resolved Rho GTPase manipulations, and a novel computational tool to study the spatiotemporal dynamics of Rho, Cdc42 and several downstream Cdc42 targets in wounded Xenopus laevis oocytes. We find that the characteristic wound-induced segregation of Rho and Cdc42 activity into concentric zones is followed by polarization of the Cdc42 zone such that Toca-1 progressively concentrates at the back of the Cdc42 zone while Arp2/3, cofilin, cortactin, and the Rho GAP p190RhoGAP progressively concentrate at the front of the Cdc42 zone, where it overlaps the Rho zone. Remarkably, the juxtaposition of Rho activity to Cdc42 is required for the polarization of p190RhoGAP, while p190RhoGAP is responsible for establishing the boundary between the Cdc42 and Rho zones. The results indicate that the characteristic segregation of the Rho and Cdc42 zones, as well as the polarization of the Cdc42 zone, arise from cortical self-organization. Further, these findings reveal a simple mechanism for hierarchical establishment of cortical patterns: recruitment of new proteins to regions of signaling compartment overlap.

Many tissues harbor quiescent stem cells that activate after injury, yet how local signals regulate this transition is not well understood. The solitary ascidian Ciona robusta provides a unique model, as bottom body fragments regenerate while upper fragments fail to do so. By comparing these regenerative and non-regenerative contexts, we reveal striking differences in transcriptional dynamics and signaling environments. Combining flow cytometry, scRNA-seq, transplantation, and fate mapping, we identified a candidate stem cell population with robust proliferative and differentiation potential following transplantation. However, regenerative capacity does not simply reflect stem cell abundance, but instead depends on region-specific signaling cues. Local expression of metabolic, immune and differentiation-related factors further underscores the importance of spatially distinct environments in shaping outcomes. Our findings show how a shared injury response can diverge into regeneration versus failure, highlighting principles that may be leveraged to enhance tissue repair in other systems.

In growth and development of plants, the signaling molecule auxin plays an instrumental role. Many patterning processes are controlled by auxin signaling in a self-organizing matter. After fertilization, such a patterning process needs to be started for the first time during plant life. Contrary to previous models, we show that the first differential auxin response that separates embryonic from extra-embryonic development in the two zygotic daughter cells happens independently of a detectable auxin gradient. Instead, differential phosphorylation of the transcriptional inhibitor Aux/IAA8 by MAP kinase signaling leads to differences in auxin sensitivity between the two daughter cells, initiating embryonic development in the apical cell. IAA8 phosphorylation in the basal cell prevents proteasomal degradation, suppressing canonical auxin responses, which initiates extra-embryonic development. MAP kinase signaling therefore precedes auxin gradients and prepatterns the apical-basal axis independently of an auxin gradient.

Cephalic neurulation and neural crest migration are defining morphogenetic events of vertebrate head development. Although traditionally viewed as independent tissue-autonomous programs, their striking spatiotemporal overlap suggests functional coupling. Here, we show that these processes are linked by reciprocal mechanochemical feedback. We find that collective neural crest migration is essential for neural tube closure. As neural crest cells delaminate and invade the surrounding mesoderm, they remodel fibronectin at the neural crest-neural plate interface, creating a specialized extracellular matrix that both separates the two tissues and promotes neural tube morphogenesis by enabling radial intercalation and apical constriction of neural plate cells. This extracellular matrix remodeling requires neural crest-specific expression of the membrane-bound metalloproteinase MMP14. Conversely, neural tube morphogenesis drives neural crest migration. Mechanical compression generated during neural plate bending induces MMP14 expression in neural crest cells, triggering extracellular matrix remodeling that feeds back to facilitate neural tube closure. Together, our findings reveal that neurulation and neural crest migration are not independent morphogenetic programs but components of a self-reinforcing mechanochemical circuit that coordinates vertebrate head morphogenesis.

Organoid models have transformed our understanding of intestinal renewal. Fluorescent imaging has been extensively used to identify key cell types and their differentiation pathways, but immunofluorescence provides only static readouts, whereas live imaging requires fluorescent-reporter engineering and is constrained by limited multiplexing and spectral overlap. Here, we introduce NuclearIDTracker, an explainable machine-learning framework that infers cell identity directly from 3D nuclear segmentations. Using a single nuclear marker, NuclearIDTracker accurately classifies intestinal cell types and integrates with single-cell tracking to resolve lineages and reconstruct dynamic state transitions during organoid development. We show that TA-like cells, rather than stem cells, drive early crypt formation and generate enterocyte and Paneth lineages, as well as the stem-cell population, which emerges only later and subsequently replenishes the TA-like compartment. Following stem-cell ablation, crypt regeneration was not driven by a single discrete cell type. Instead, multiple epithelial populations converged on a proliferative regenerative state with a nuclear phenotypic signature that resembled, but remained distinct from, that of homeostatic TA-like cells, and a YAP/TAZ-associated fetal-like transcriptional signature. Thus, nuclear phenotypic signatures resolve cell identity and reveal coordinated epithelial plasticity during crypt regeneration. NuclearIDTracker establishes a non-perturbative tool to quantify cell identity and state dynamics at single-cell resolution, revealing previously inaccessible biological dynamics and expanding the toolkit for studying epithelial homeostasis, regeneration, and disease.

Lymphatic dysfunction drives severe and often intractable human diseases, yet the cellular mechanisms that establish functional lymphatic vasculature remain poorly understood. In the intestine, lacteals are specialized lymphatic vessels that absorb dietary lipids and rely on surrounding villus smooth muscle to propel lymph, forming the muscular-lacteal complex (MLC). How distinct mesenchymal populations coordinate assembly of this functional lymphatic unit remains unknown. By integrating developmental single-cell profiling, genetic lineage tracing, conditional mouse genetics, and functional assays of lipid absorption, we identify Notch3 as a central organizer of MLC development that coordinates communication between distinct mesenchymal lineages. While Notch3 promotes smooth muscle differentiation within the PDGFR lineage, PDGFR{beta} lineage cells do not directly contribute to villus smooth muscle. Instead, they function as Notch3-dependent signaling hubs that instruct expansion and differentiation of neighboring PDGFR smooth muscle progenitors via paracrine TGF{beta} signaling. Loss of Notch3 in PDGFR{beta} cells disrupts MLC development, impairs intestinal lipid absorption, and causes postnatal growth failure and lethality. Restoration of TGF{beta} signaling rescues the structural, functional, and survival defects caused by Notch3 loss, identifying TGF{beta} as a critical downstream effector of the Notch3 pathway. Furthermore, selective inhibition of canonical Notch signaling in the PDGFR{beta} lineage fails to phenocopy Notch3 deletion, revealing a non-canonical mechanism of Notch3 function in intestinal mesenchymal development. Together, these findings establish PDGFR{beta} cells as essential mesenchymal signaling organizers and define a new paradigm in which lineage-specific, non-canonical Notch3 signaling coordinates villus stromal communication to build a functional intestinal lymphatic niche.

Lifelong stem cells are maintained by a cellular microenvironment called the niche, which enables tissue homeostasis. Proper niche construction is essential for persistent function, but studying niche formation is challenged by the inaccessibility of most niches to in vivo visualization during development. Innovations imaging the Drosophila testis are now allowing investigation of niche inception. F-actin polarizes to precise cell interfaces during testis niche assembly. Yet it is unknown whether polarization directs niche cell motility, or reflects adhesive sorting in response to formation of niche cell contacts. By adapting a method to optogenetically manipulate cortical F-actin via disruption of Rho1, we interrogate the role for cytoskeletal polarization during niche formation with tissue and temporal specificity. Rho1-mediated disruption of F-actin polarization caused defects in niche anterior assembly and architecture. Also, fewer cells adopted bona fide niche identity, given diminished Fas3, N-Cadherin, and Islet. These disrupted niches fail in signaling to germ cells to establish stem cell identity. We reveal that polarized F-actin is crucial for establishing cell contacts to form a functional niche, and to maintain cell identity in the developing tissue. Summary StatementOptogenetic cortical localization of cytoskeletal disruptors reveals tissue and temporal specific requirements for F-actin polarization in establishing a functional stem cell niche.

The release of extracellular vesicles (EV) from the primary cilium is a conserved process observed in many cell types. It serves as a rapid and efficient mechanism to release select proteins from the cilium, which can be used for either intercellular communication or membrane material disposal. Previous studies have shown that the release of EVs from the cilium relies on the actin cytoskeleton and proposed several molecular mechanisms that may perform this function. Using the model of IMCD3 cells, we now demonstrate that this process relies on actomyosin contractility supported by non-muscle myosin IIA acting downstream of the RhoA-ROCK signaling pathway. We further showed that the cilia of these cells release EVs independently of de novo actin polymerization, which we confirmed using an in vivo model of mutant photoreceptor cells that release massive amounts of vesicles from their cilia instead of elaborating into light-sensitive outer segment membrane structures.

ANGPT2 is widely recognized as a critical regulator of pathological neovascularization. By analyzing scRNA-seq data from neonatal retinas, we demonstrate that Angpt2 transcripts are highly enriched in tip cells relative to other endothelial subtypes, where Angpt1/4 expression is absent. However, mechanisms underlying ANGPT2 function at angiogenic fronts remain inadequately understood. Here, we show that endothelial Angpt2 deletion severely disrupted retinal vascularization, characterized by neovascular tufts and micro-hemorrhage. Similar angiogenic defects also occurred in the brain, but were less evident in other tissues examined. Mechanistically, ANGPT2 insufficiency attenuated retinal tip cell invasion with aberrant mural cell coverage, compromising sprouting into non-vascularized tissues. Retinal RNA-seq analysis revealed that transcripts associated with endothelial migration and junction assembly were reduced in Angpt2 mutants compared to littermate controls, while upregulated genes were enriched in hypoxia-responsive pathways and mural cell development. Notably, abnormal mural-tip cell associations were detected within 48 hours post-Angpt2 deletion, displaying also a hypoxia-driven transcriptomic signature. These closely resemble the vascular pathologies observed in human retinopathy of prematurity. In contrast, Angpt1 insufficiency or Angpt4 deficiency primarily affected venous morphogenesis. Collectively, our findings imply that ANGPT2 is essential for driving tip cell invasion during sprouting angiogenesis, and that its insufficiency triggers hypoxia-driven vascular anomalies.

Viable yet suboptimal ("loser") cells can be recognized and selectively eliminated when in the presence of neighboring fitter ("winner") cells through cell competition, thereby promoting optimal tissue fitness and homeostasis. One mechanism through which cells compare their relative fitness levels relies on isoforms of the conserved transmembrane protein Flower (Fwe). Despite the role of Fwe-dependent cell selection in several pathophysiological processes, little is known about downstream components of this pathway. In this study, we develop a versatile clonal interaction assay in Drosophila, the Easy Win Assay. By overexpressing human FWE1 Lose isoform to trigger cell competition, we perform an unbiased whole-genome RNAi screen and identify new pathway modulators. We show that the receptor Grindelwald/TNFR is necessary in either winner or loser cells to drive Eiger/TNF--independent elimination of losers, whereas the scaffolding protein Veli/LIN-7 is simultaneously required in both cell populations. We further demonstrate that Fwe, Grindelwald and Veli redistribute to establish a previously undescribed bilateral communication module at winner-loser interfaces, promoting intercellular communication and elimination of loser cells. These findings show that distinct cell elimination pathways converge on a common execution module while remaining independently regulated upstream, enabling flexible recognition and elimination of diverse damaged or dangerous cells.