Developmental Cell

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.

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.

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.

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.

Planar cell polarity (PCP) in epithelia is characterized by the polarized distribution of two opposing, membrane-associated PCP complexes across cell junctions. Transmembrane components of the PCP complex bridge cell junctions and organize into punctate, intercellular assemblies that exhibit a high degree of stability. Here, we define the contributions of the cytoplasmic PCP protein, Dishevelled (Dvl), in the sub-micron scale organization and stability of PCP complexes. Using endogenously-tagged fluorescent PCP reporters in the embryonic mouse epidermis, we quantify PCP protein mobility and clustering during polarization. We find that as transmembrane proteins immobilize into puncta, Dishevelled (Dvl2/3) co-accumulates with its transmembrane partner Frizzled (Fz6) in a polarized manner and stabilizes clusters of PCP complexes. We identify a previously unknown function for the oligomerizing DIX domain of Dvl3, typically associated with Wnt signaling, in Dvl3 asymmetric localization. These observations underscore a role for Dvl oligomerization in assembly and stabilization of asymmetric PCP puncta.

Brown algae evolved independently from animals, land plants and other algae, and we know very little about the spatio-temporal dynamics of their embryogenesis. Here, we used time-lapse, bright-field microscopy to study cell division and lineage development during early embryogenesis in the kelp Saccharina latissima, a large brown alga. We discovered a radical change of cell identity as early as the 4-cell stage: after fertilization, the zygote underwent two or three unequal cell divisions before the basal cell - that closest to the maternal tissue - stopped dividing and radically differentiated into a hyperpolarized cell, the rhizoid, which anchors the embryo to the substrate. RNA-seq analysis showed that differentiation of rhizoid cells was preceded by expression of 130 basal cell-specific genes. Phylostratigraphic analysis further revealed that more than 40% of these basal cell-specific genes appeared after the emergence of the brown algae group, and their functions are largely unknown. By contrast, the apical cell predominantly expressed more ancestral, metabolism-related genes, and it continued to divide to produce the long, blade-shaped thallus of the alga. The early and radical nature of cell differentiation in Saccharina embryos, combined with differential gene expression from various evolutionary periods, highlights the unique mechanisms of embryogenesis of this alga.

How spatial patterns arise during embryonic development is classically explained by the French Flag model, in which cells acquire positional identities by interpreting morphogen concentration thresholds. However, in many developmental systems, spatial patterns instead emerge progressively through temporal programs of gene expression that are transformed into spatial organization. In the short-germ insect Tribolium castaneum, both periodic pair-rule gene expressions that generate body segments and non-periodic gap gene expressions that establish regional identities arise sequentially at the posterior and propagate anteriorly in waves across the developing embryo. Understanding how such temporal gene expression programs are translated into spatial patterns remains a major challenge. To address this problem, we developed a sequential multiplexed imaging strategy based on hybridization chain reaction (HCR), enabling visualization of up to ten anterior-posterior (AP) patterning genes within the same embryo. By combining this approach with intronic-exonic labeling, we established a framework to infer gene expression dynamics and propagatory behavior during AP patterning. Using this framework, we show that gap gene expression domains remain dynamic and continue to propagate during tissue elongation, indicating that spatial patterns are actively remodeled throughout development. We then directly compared temporal gene activation at the posterior with the resulting spatial organization of pair-rule and gap genes. Surprisingly, while primary pair-rule genes preserve their temporal phase relationships in space, gap genes do not. Instead, the relative positioning of gap gene domains progressively changes as they move anteriorly, indicating that the final spatial organization of gap genes is actively reshaped during propagation rather than being directly inherited from the initial temporal sequence. The continued propagatory behavior of gap gene domains suggests that such reshaping could arise through differential propagation dynamics between genes and/or through progressive reconfiguration of underlying gene regulatory interactions during pattern formation. Together, these findings reveal that temporal-to-spatial patterning can involve active transformation of temporal information rather than a simple mapping from time into space.

Directed cell migration is a vital process that depends on the combined activities of attractive and repulsive cues. As it is essential for normal development, the precise identity of guidance signals and the underlying molecular and cellular mechanisms is being rigorously investigated. In a Drosophila embryo, PGC migration is orchestrated by non-cell autonomous repulsive and attractive cues, controlled by Wunen(s) - Wunen and Wunen2 and, HMGCoA-reductase (Hmgcr), respectively. Hedgehog (Hh), a PGC attractant, is potentiated by Hmgcr. We demonstrate that Wunen(s) employ both nonautonomous and autonomous modes to inhibit Hh signaling. Consistently, in embryos maternally compromised for wunen, mesodermal cells and PGCs accumulate excess Hh, leading to precocious clumping of the PGCs. This behaviour is reminiscent of PGC-specific loss of patched (ptc) - the Hh receptor and an antagonist of Smoothened (Smo), a G protein-coupled receptor (GPCR), involved in Hh signal transduction. Consistently, Wunen(s) inhibit membrane localization of Smo. Conversely, simultaneous overexpression of wunen mitigates PGC scattering induced by ectopic hmgcr expression. Finally, unbiased lipidomics of embryonic extracts after maternal knockdown of wunen confirms disruptions in lipid metabolism. We discuss the mechanistic underpinnings of Wunen(s) involvement in repressing Hh signalling to engineer PGC migration.

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 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.

Muscle loading is required for embryonic tendon growth; however, the underlying mechanisms that regulate tendon development downstream of mechanical cues remain unidentified. Although tendons in muscle paralysis models are structurally and functionally inferior, whether these differences arise from cell or matrix deficits remains unclear. Analysis of muscular dysgenesis embryos by atomic force microscopy showed that structural and functional deficits in paralyzed tendon arise in part from reduced proliferation and collagen fibril disorganization. Bulk and single cell transcriptional analyses reveal that both collagenous and non-collagenous extracellular matrix components, as well as cytoskeletal and actomyosin-associated proteins, are dysregulated in mdg tendons, whereas tendon markers remain unchanged. Surprisingly, we find that an arrest of TGF{beta} signaling occurs during normal embryonic tendon growth and that TGF{beta} signaling is abnormally prolonged in paralyzed embryos. We also show for the first time, that specification of the epitenon depends on muscle contraction. Together, these findings establish cell and molecular requirements for muscle contraction in embryonic tendon development. TeaserMuscle contraction is required for embryonic tendon development through regulation of TGF{beta} signaling, epitenon formation, and matrix organization.

Integrin-linked kinase (ILK) and kindlin-2 (K2) are key components of focal adhesions (FAs) that regulate cell-matrix adhesion and integrin signaling. Both proteins directly bind each other, but how they influence each others localization to FAs and binding to integrins remains a subject of ongoing debate. Here, we establish a sensitive workflow to study protein-protein interactions in cells by combining methods from biochemistry, cell biology and super-resolution microscopy. Together with an analytical framework this approach allowed us to distinguish direct from indirect molecular interactions and construct detailed interaction networks. Disrupting the ILK-K2 interaction reduced ILK localization to FAs and compromised integrin function, whereas K2 recruitment was unaffected. Our interdisciplinary approach also revealed that ILK does not directly bind {beta}1-integrin cytosolic domains in vitro and in cells. Instead, ILK was recruited to integrins exclusively through a K2-dependent mechanism, primarily via K2 bridging ILK and {beta}1 integrins. These findings define the hierarchical relationship between ILK and K2 in FAs and highlight the essential role of K2-mediated ILK recruitment for integrin adhesion and signaling. Significance StatementHow cells anchor to their environment is a fundamental question in biology. Integrins provide such a connection by bridging the extracellular matrix and the cytoskeleton. A central regulator of the integrin machine is Integrin-linked kinase (ILK). How ILK is recruited to {beta}1 integrins is hotly debated since its discovery more than 30 years ago. By integrating cell biology and biochemistry with super-resolution DNA-PAINT microscopy and a novel spatial analysis framework, we demonstrate that ILK does not bind directly to integrin cytoplasmic tails. Instead, we found that ILK is recruited by kindlin-2 (K2) to active, adhesion plaque-resident integrins. This work resolves a long-standing controversy in cell biology and establishes a versatile workflow for distinguishing direct from indirect protein-protein interactions in situ.

Despite recent explorations of tissue-level epithelial morphogenesis, how the embryonic brain wall, an epithelial derivative unique in its extensive cellular stratification coupled with epithelial-cell heightening to [~]0.5 mm, achieves thickening is unknown. Furthermore, the role of the inner curvature of the wall in this process remains unclear. Thus, whether the apically concave dorsal cerebral (pallial) wall thickens inward, not only outward as previously thought, was examined in culture and in vivo. The pallial wall in the midembryonic period, but not the earlier pallial wall, thickened inward at a pace equivalent to that of neuronal accumulation, which was found via stress-release tests to proceed in a compressive manner. The inhibition of actomyosin-mediated contraction of the inner/apical surface prevented the pallium from thickening, while inducing apical-surface buckling, suggesting the necessity of this inward thickening to actively avoid overcrowding. In contrast, inward bulging of the apically convex ganglionic eminence occurs more passively via pushing of the low-actomyosin apical surface by the constituent cells.