Developmental Cell

Organs are composed of a complex arrangement of diverse cell types that can originate from independent cell lineages or shared progenitors. Skeletal muscle is derived from mesodermal Pax7+ stem/progenitor cells that can differentiate into myoblasts to form muscle fibers. During embryogenesis, however, somitic Pax7+ cells can also give rise to non-muscle cell types, including dermis and adipocytes. Here, we asked whether Pax7+ cells retain such multipotency during early postnatal growth of limb muscles. Using lineage tracing, we uncovered unexpected plasticity at early postnatal days, leading to the generation of multiple non-myogenic lineages, including a previously unrecognized Pax7-derived subpopulation of fibro-adipogenic progenitors that we termed Pax7FAPs and further investigated. Using mouse models, we further show that Notch signaling primes neonatal Pax7+ cells toward a fibrogenic molecular identity at the expense of myogenic differentiation, thereby biasing their trajectory toward a fibrogenic fate. In the adult muscle, long-term tracing revealed that neonatally produced Pax7FAPs persist into adulthood. In addition, injury in adult muscle triggered de novo generation of Pax7FAPs, which displayed higher proliferative capacity than resident stromal cells. This newfound multipotency of postnatal Pax7+ cells adds a new dimension to our understanding of cellular contributions during postnatal muscle development and regeneration.

HighlightsO_LILamin-A and lamin-B1 are essential for midgestational embryogenesis. C_LIO_LILamin-A/B1 are required for proper yolk sac endoderm (YSE) gene regulation. C_LIO_LILamin-A/B1 maintain LADs organization and chromatin interactions in YSE. C_LIO_LILamin-A/B1 and YSE transcription factors support proper YSE gene expression. C_LI Lamins are intermediate filament proteins functioning as ubiquitous structural components of the nuclear lamina that interact with and organize the Lamina-Associated chromatin Domains (LADs). LADs remodel during development and lamins maintain LADs and gene expression profile specific to a given cell type. How ubiquitous lamins achieve cell-type-specific functions during development remains unknown. We show lamin-A and -B1 are required for mouse midgestational embryogenesis and maintain LADs, 3D chromatin interactions, and gene expression in the yolk sac endoderm (YSE). Both lamin-regulated genes and remodeled LADs in YSE cells contain binding motifs of YSE-relevant transcription factors. By analyzing changes in chromatin interactions upon lamin-A and -B1 knockout, we reveal that chromatin neighborhoods maintained by these lamins can influence gene expression orchestrated by YSE-relevant transcription factors. Our findings explain how the ubiquitously expressed lamins can collaborate with lineage-relevant transcription factors to maintain LADs and gene expression programs in specific cell types.

How regenerative capacity originates during development remains poorly understood, even in vertebrates with exceptional adult regenerative ability. Using the axolotl, we identify a defined embryonic window between stages 30 and 34 during which the tail region transitions from a regeneration-incompetent to a regeneration-competent state. Amputations across staged embryos reveal that earlier embryos entirely fail to regenerate, whereas later embryos regenerate functional tails. Notably, tail stumps from nonregenerating embryos can recover the ability to regenerate when reamputated at later stages, demonstrating that early regenerative failure does not permanently impair regenerative capacity. This differs from the transient refractory period described in Xenopus, where regenerative competence is lost and reacquired around the end of tail outgrowth, and indicates that staged acquisition of regenerative competence is a broadly shared but mechanistically distinct feature of amphibian development. To determine whether this transition reflects changes in progenitor composition, we analysed the single-cell transcriptional landscapes of axolotl tail buds across this window. Tail bud progenitors, including neuromesodermal progenitors, persist through the transition, indicating that the onset of regenerative competence is unlikely to be explained by the loss of embryonic progenitors. Finally, using Tbxt (Brachyury) crispant axolotls with severe axial defects, we show that tail regeneration occurs effectively despite earlier abnormal embryonic tail development, with functional uncoupling of the mechanisms of tail development and regeneration. This framework provides new opportunities for identifying the drivers of regenerative competence and understand why this capacity is lost in other vertebrate species.

Adult zebrafish possess a remarkable ability to regenerate their heart following cardiac injury. Over the past decades, our understanding of the diverse cell types involved in zebrafish cardiac regeneration has greatly advanced. However, the mechanisms governing their interaction and how heterocellular crosstalk drives regeneration remain poorly understood. Here, we identify cardiomyocyte autophagy as a key link between the cardiomyocyte injury response and heterocellular crosstalk between cardiomyocytes and macrophages. We find that cardiomyocyte autophagy is downstream of AP-1 transcription factors. Using newly generated genetic tools, we find that cardiomyocyte autophagy is an important regulator of cardiomyocyte protrusion into the fibrotic injured tissue and its disruption leads to defects in scar resolution. Notably, we find that blocking cardiomyocyte autophagy has a marked effect on the transcriptomic signatures in cardiac macrophages, shifting gene expression from phagocytic/pro-inflammatory/pro-reparative towards pro-angiogenic and pro-fibrotic states. Altogether, our results uncover autophagy as a mechanism linking cardiomyocyte injury responses to macrophage phenotype and coordinated tissue remodeling during heart regeneration.

Specification of the inner cell mass (ICM) and trophectoderm (TE) at the first mammalian cell fate decision requires the transcription factor Tead4, yet what restricts Tead4 activity to presumptive TE cells remains unknown. Tead4 localizes to mitochondria, and the ICM and TE harbor distinct mitochondrial populations, but whether Tead4 distribution varies across mitochondrial subtypes in the cleavage-stage embryo has not been examined. Here we used fluorescence-activated mitochondrial sorting (FAMS) to characterize mitochondrial subpopulations in mouse metaphase-II oocytes and 8-cell embryos with respect to size, mitochondrial membrane potential ({Delta}{Psi}m), and Tead4 protein content. Mitochondria are heterogeneous in size and {Delta}{Psi}m in both developmental stages, with large mitochondria exhibiting markedly higher {Delta}{Psi}m than small mitochondria. Tead4 protein is concentrated in the large, high-{Delta}{Psi}m mitochondrial subpopulation in 8-cell embryos, with 75% of large mitochondria containing Tead4 compared to only 3% of small mitochondria. The overall size distribution of the mitochondrial pool is maintained between oocytes and 8-cell embryos; Tead4 accumulation within the large mitochondrial fraction is therefore a developmentally regulated process initiated specifically during the early embryogenesis. These findings establish for the first time that Tead4 localizes preferentially to large, high-{Delta}{Psi}m mitochondria in the cleavage-stage embryo, providing a previously unrecognized cellular basis for understanding how Tead4 bioavailability may be regulated prior to TE specification.

The discovery of neuromesodermal progenitors (NMPs) -- a bipotent progenitor population in the tailbud that gives rise to traditionally ectodermal and mesodermal tissues -- has disrupted the classical view that progenitors of the three distinct germ layers are exclusively segregated during gastrulation. However, until now the notion of lineage restriction of the endoderm to traditional gastrointestinal and respiratory tissues has largely remained intact. Here, we describe our discovery of a unique subpopulation in the chick endoderm that initially lines the ventral surface of the posterior organizer (Hensens node), but at the trunk-to-tail developmental switch, undergoes an FGF-dependent epithelial-to-mesenchymal transition, invading the tailbud and subsequently differentiating into a remarkably broad range of cell types including somites, notochord, and neural tube. Strikingly, ablation of this endodermal cell population results in a severe ([~]50%) reduction in axis elongation rate. Through single cell RNA sequencing and in situ hybridization chain reaction, we conclude that these cells lose their endodermal identity upon ingression, giving rise to NMPs that are biased toward mesodermal fates. Lineage tracing reveals that the node endoderm harbors a mixed multipotent population of progenitor cells capable of generating progeny that span endoderm and mesoderm or endoderm and ectoderm. These findings illustrate a previously unappreciated endodermal source of NMPs, and further demonstrates the breakdown of traditional lineage restriction of germ layers in the posterior embryo.

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.

The formation of functional corneal endothelial cells during development requires tight coordination between tissue-scale growth and cell-scale organization, yet how these processes are integrated in three dimensions remains poorly understood. Here, we combine high-resolution confocal imaging with quantitative analysis to reconstruct the morphogenesis of the chick corneal endothelium across embryonic development. Using a pipeline integrating 3D nuclear segmentation, Voronoi-based topological mapping, and spatial statistics, we link macroscopic globe expansion to single-cell geometry and lattice organization. We identify a multiphasic relationship between tissue growth and cell density, driven by temporal decoupling of organ expansion and proliferation. During early development, rapid globe expansion induces cellular stretching and spatial heterogeneity, followed by a phase of density accumulation and geometric refinement. Despite these dynamic conditions, the endothelial sheet maintains a robust monolayer architecture with minimal z-axis stratification. Quantitative topological analysis reveals that hexagonal packing is preserved from early stages and progressively refined through reduction of area variability and spatial clustering. Nearest-neighbor and Clark-Evans analyses demonstrate a transition from localized clustering to a more uniform spatial distribution, consistent with increasing packing regularity. Transient out-of-plane deviations coincide with key mechanical transitions, suggesting a role for 3D remodeling in accommodating mechanical stress. Concomitantly, junctional and cytoskeletal organization undergo progressive maturation. N-cadherin is established early at cell-cell interfaces, while Zonula Occludens-1 (ZO-1) transitions from diffuse localization to apically enriched tight junctions aligned with cortical actin. In parallel, microtubule organization becomes increasingly polarized to the apical domain, coinciding with the emergence of primary cilia. Together, these changes reflect coordinated establishment of epithelial polarity, barrier function, and mechanical stability. Overall, our study provides a multiscale, imaging-driven framework for understanding how epithelial tissues achieve and maintain geometric order under mechanical strain, establishing the corneal endothelium as an exemplar for linking developmental mechanics, 3D architecture, and epithelial topology. Summary StatementUsing 3D imaging and quantitative analysis, this work reveals how corneal endothelial cells stay organized and form a regular pattern during growth, despite ongoing changes in tissue size and shape.

The neuronal endoplasmic reticulum (ER) extends from the soma into axons and dendrites to coordinate protein trafficking, lipid metabolism, inter-organelle organization, and calcium homeostasis. Conserved genes involved in shaping the tubular ER are implicated in neurodevelopment and neurodegeneration, suggesting that ER structure and dynamics influence neuronal health and drive pathogenesis. However, the links between ER morphology and neuronal function and resilience remain incompletely understood. While models typically depict the neuronal ER as a fully continuous network, here we show that micron-scale ER discontinuities in neurites are unexpectedly common in young, unstressed C. elegans. These discontinuities occur in both axonal and dendritic compartments with a consistent frequency that varies between motor and mechanosensory neuron types. Using live imaging and photokinetic assays of endogenously tagged markers of the ER, we confirm that these gaps reflect true loss of ultrastructural continuity. Subpopulations of ER tubule tips are highly motile, and the majority of ER discontinuities are resolved in less than an hour. Suggesting the formation of discontinuities is linked to cellular damage, their frequency increases with both age and environmental stress. Finally, in agreement with prior observations across models, discontinuities are exacerbated by impairment of certain ER shaping factors involved in hereditary spastic paraplegia, such as reticulon. These findings reveal a model where ER discontinuities are not uncommon in healthy animals, and provide a tractable system in C. elegans to dissect the molecular mechanisms maintaining ER structural homeostasis in vivo.

Thyroid hormone (TH) is a systemic regulator of vertebrate development, yet its role in the maturation of the stratified skin remains poorly defined. Using the skin at the edge of the zebrafish caudal fin, we defined the trajectory of epidermal maturation during the transition from juvenile to adult. We found the peripheral edge (PE) of the fin exhibits positive allometric expansion that is dependent on TH: in thyroid-ablated, hypothyroid backgrounds, the growth of the PE is limited. We showed that TH drives normal PE growth by stimulating both cell proliferation and hypertrophy. Further, we demonstrated that TH acts upstream of the Notch pathway to regulate growth of the PE. While TH signaling machinery is broadly expressed throughout the fin, Notch pathway activation is localized and highly enriched in the PE. Repressing Notch activity prevented PE expansion, while upregulating Notch in a hypothyroid background was sufficient to increase hypertrophy and partially rescue PE expansion. By identifying Notch as a region-specific effector of TH-driven hypertrophy, our findings show a mechanism by which systemic endocrine signals are translated into local tissue morphogenesis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/713269v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1381159org.highwire.dtl.DTLVardef@1fb2421org.highwire.dtl.DTLVardef@100fed3org.highwire.dtl.DTLVardef@5a0480_HPS_FORMAT_FIGEXP M_FIG C_FIG

Left-right asymmetry in vertebrate embryos is established by the left-right organizer (LRO), with the zebrafish Kupffers vesicle (KV) providing a tractable model for studying de novo epithelial morphogenesis. During KV formation, dorsal forerunner cells (DFCs) initially form polarized attachments to the enveloping layer (EVL) before reorganizing into multicellular rosettes that precede lumen formation. Here, we show that while DFC-EVL junctions form independently of mitosis, early cytokinetic events play an instructive role in remodeling these contacts. Live imaging and targeted laser ablation reveal that cytokinetic bridges and their associated microtubule bundles recruit actin, seed rosette centers, and promote the transition from external to internal epithelial organization. Disruption of early, but not later, DFC divisions impairs actin accumulation, rosette coalescence, KV detachment from the EVL, and lumenogenesis. These findings identify a temporally restricted role for cytokinesis in organizing cytoskeletal architecture and reveal how division history directs epithelial tissue assembly during LRO development.

Mitochondrial quality control (MQC) is essential for retinal homeostasis, yet how distinct mitophagy pathways are coordinated within specialized retinal cell types remains poorly understood. Here, we show that Muller glia engage distinct mitophagy programmes that are differentially activated across physiological, metabolic stress, and differentiation contexts. Using pathway-resolved analyses supported by mouse and human single-cell transcriptomic datasets, we demonstrate that PINK1-dependent and receptor-mediated mitophagy pathways coexist within Muller glia and exhibit distinct functional and spatial regulation. To enable precise, time-resolved interrogation of these processes, we developed MQ-MG2, a spontaneously immortalised Muller glial model stably expressing the Mito-QC reporter while preserving endogenous mitophagy adaptors and metabolic features of primary Muller cells. Using this system, we identify context-dependent activation of mitophagy pathways with spatial relevance in vivo and reveal transient coordination of PINK1-dependent and receptor-associated mitophagy during Muller glial neurogenic differentiation. Suppression of fission-dependent mitophagy impaired the acquisition of complex neurite features in MQ-MG2, with a comparable phenotype observed following targeted PINK1 deletion in human neurogenic cells. Together, these findings position Muller glia as active integrators of mitochondrial quality control, capable of engaging distinct mitophagy programmes according to cellular context.

Ribosome biogenesis is a conserved and highly regulated process that starts in the nucleolus, a membrane-less multi-phase organelle. Although the architecture of the nucleolus is known to change due to perturbations, how nucleolar organization is modulated during physiological processes to meet changing translational demands remains unclear. Here, we use zebrafish oogenesis as a developmental context requiring a rapid expansion of translational capacity to investigate the regulation of nucleolar architecture. We show nucleoli undergo coordinated changes in number, size, subnuclear localization, and layering throughout oogenesis. We further demonstrate that nucleoli form around extrachromosomal DNA circles that contain the rDNA locus. Notably, mouse oocytes undergo similar developmental changes in nucleolar layering and phase organization, indicating that remodeling of nucleolar condensates is a conserved feature of oogenesis. These findings reveal previously unexplored regulation of nucleolar architecture as developmental adaptations to changing biosynthetic needs.

In developing tissues, cells differentiate into distinct cell types and form complex spatial patterns. How distinct patterning systems interact during tissue growth to shape tissue composition and spatial organization remains poorly understood. Here, we investigate this question in the abaxial leaf epidermis of Arabidopsis thaliana, in which the same pool of progenitor cells gives rise to stomata, pavement cells, and giant cells. Using a quantitative approach combining Euclidean and network-based spatial analysis, we show that stomatal number and density are robust to reduced endoreduplication, whereas forced endoreduplication actively competes with the stomatal lineage to reduce stomatal number. Furthermore, we show that the stomatal spatial pattern is also shaped by the broader tissue context such as cell growth, cell division, and giant cell patterning, with distinct consequences for stomatal spatial distribution and cellular arrangement. Our results highlight that the interplay between patterning systems must be considered to understand how tissue organization is established.

Morphogenesis often requires different cell types to coordinate their behaviors for an harmonious developpement. How these different cell type behaviors are synchronized within and between tissues remains one of the important questions to fully understand morphogenesis. We used the zebrafish developing spinal cord to study this question. At later stages of neurogenesis, the lumen of the neural tube remodels, by reducing its height dramatically to form the persisting ventral central canal, a morphogenetic process conserved in vertebrates. By combining genetics, cell signaling manipulation with antagonist drugs and high-resolution in vivo live imaging, we better characterised the dynamics and control of this remodeling process. We showed that the lumen retraction depends on Gli activity regulation, a downstream effector of the Shh morphogen signal. We further established that the lumen retraction is instrumental in the cellular elongation of spinal roof plate cells, a population that forms the ceiling of the spinal cord lumen. Our work therefore establishes that the Gli transcriptional regulators under the control of long-range morphogen Shh control lumen retraction and that this retraction is a key driver of the roof plate cells extension.

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.

Branched tubular organs represent a common solution to the problem of fluid transport in large animals. The growth of new branches has been extensively studied but the regulation of branch pruning remains underexplored. Here, we investigate branch removal in the stereotyped branching patterns of the developing Drosophila airways. After progenitor invagination, the tips of the distal airways generate a stereotyped branching pattern in each central metameric unit. An intriguing exception in the repeating patterns is the lack of branches targeting the visceral mesoderm (visceral primary branch, VB) in the third and nineth metameres. We show that these branches initially form but the localized expression of the pro-apoptotic gene reaper and resultant apoptosis prune them. We reveal that VB3/9 pruning entails four sequential programs. First, a common distal outgrowth program promotes budding and extension of the primary branches, including VB3 and 9. Second, a VB identity program is established representing the ground state of all primary branches. Third, the Bithorax-Complex transcription factors define metameric identities and interfere with the VB program to induce reaper and apoptosis specifically in VB3/9 in a concentration dependent manner. Finally, the default VB cell identity is transformed by extrinsic BMP/Decapentaplegic and WNT/Wingless into more derived primary branch identities and spared from pruning in metamers 3 and 9. Our results demonstrate that molecular and genetic circuits promoting branch emergence and extension can be regionally modified and deployed also for branch pruning. Author summaryMany of our internal organs like the lung, kidney and various glands are composed of epithelial tubes. The function of these organs is sustained by the morphologies of the constituting tubes. The formation of distinct branching patterns in tubular networks including regional variegations can be accomplished by branch ramification and branch pruning. Extensive studies of tube ramification and extension showed that they are typically regulated by extrinsic guidance cues. In contrast to branch extension, branch maintenance and removal is much less understood. Here, we identify a program that intrinsically promotes apoptosis to prune specific branches in the developing Drosophila airways. Intriguingly, the branch extension program is diverted to a branch pruning program. Activation of the branch outgrowth program is prerequisite for both branch extension and pruning. The Hox gene expression code that characterizes regional cell identities along the body axes intrinsically interferes with the branch extension program of visceral primary branches (VB) targeting the visceral muscles. This results in VB pruning only in metameres 3 and 9, where extrinsic WNT and BMP guidance cues direct towards distal branch identities that are spared from apoptotic pruning. Similar interplay of branching and pruning programs may operate in sculpting our lung and vasculature.

Adherens junctions (AJs) undergo dynamic remodelling during epithelial morphogenesis, requiring precise coordination between adhesive proteins, intracellular adaptors, and cytoskeletal regulators. In addition to cadherins, which mediate core cell-cell adhesion and connect junctions to the actin cytoskeleton, other adhesion molecules from the immunoglobulin superfamily (IgCAM) also contribute to AJ organisation. In Drosophila, Echinoid (Ed), a nectin-like IgCAM, localises along the entire AJ, whereas Sidekick (Sdk), another IgCAM, is predominantly enriched at tricellular adherens junctions (tAJs). Although both proteins interact with overlapping intracellular partners, how they functionally relate to one another has remained unclear. Here, we investigate the spatial, molecular, and functional interplay between Sdk and Ed during embryonic epithelial morphogenesis. Using SRRF-imaging we show that Sdk and Ed frequently colocalise at tAJs but also display adjacent or spatially separated distributions; together with proximity-labelling experiments, these results suggest that Sdk and Ed engage in transient and dynamic associations rather than forming a stable complex. Functional analyses reveal that they influence each others accumulation, indicating bidirectional regulatory interactions. We find that Sdk modulates Ed levels along the entire AJs and affects Ed enrichment at tAJs. We provide evidence that this regulation involves changes in Ed intracellular trafficking, suggesting that Sdk modulates Ed levels at AJs at least in part by controlling its trafficking. Genetic analyses uncover previously unreported contributions of ed to tracheal cell intercalation and of sdk to dorsal closure, and reveal strong genetic interactions between the two genes, indicating cooperative yet context-dependent functions. Consistent with this, we find that Sdk and Ed converge on shared intracellular adaptor proteins, including Canoe and Polychaetoid, modulating their levels and junctional enrichment. Together, our findings support a model in which dynamic, multi-component protein complexes assemble at bicellular and tricellular AJs, integrating shared and junction-enriched components that engage in multiple, simultaneous, and mutually influencing interactions. This interconnected network would confer the spatiotemporal robustness and flexibility required to support the distinct cellular behaviours underlying tissue-specific remodelling.

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.

Heart regeneration requires coordinated immune activation, timely inflammatory resolution, and dynamic extracellular matrix (ECM) remodeling in addition to cardiomyocyte (CM) proliferation. However, the cytokine signals that instruct immune cell functions during cardiac repair remain incompletely understood. Here, we identify interferon-gamma (IFN-{gamma}) as a critical regulator of macrophage plasticity in zebrafish heart regeneration. IFN-{gamma} signaling components are dynamically activated following cardiac injury, with early induction of ifng1 and temporally coordinated receptor expression. Genetic ablation of ifng1 impairs myocardial regeneration, resulting in reduced CM proliferation and persistent fibrotic scarring. Temporal transcriptional profiling reveals sustained inflammatory signatures, impaired efferocytosis, and abolished reparative programs, accompanied by aberrant immune cell dynamics and retention of injury-derived debris in mutant hearts. Transcriptomic analysis of cardiac macrophages further reveals that IFN-{gamma} deficiency disrupts the transition from an inflammatory state to a reparative, ECM-remodeling phenotype, leading to reduced collagen denaturation and diminished CM protrusion at the injury border zone. Inducible- and macrophage-specific blockade of IFN-{gamma} signaling phenocopies defects in global knockout, establishing a cell-autonomous requirement for IFN-{gamma} in coordinating regenerative immune function. Collectively, our findings define an IFN-{gamma}-dependent macrophage reprogramming axis that couples inflammatory resolution to ECM remodeling in heart regeneration, elucidating how cytokine signaling actively instructs tissue repair. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/712551v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@cefbecorg.highwire.dtl.DTLVardef@fd56dborg.highwire.dtl.DTLVardef@517495org.highwire.dtl.DTLVardef@1bd0851_HPS_FORMAT_FIGEXP M_FIG C_FIG