Cells in vivo experience mechanically diverse microenvironments in which physical confinement is a pervasive but poorly understood regulator of their behavior and fate. Whether and how mechanical confinement governs immune cell differentiation remains unknown. Here, we reveal that a mechanical cue-long-term confinement is sufficient to drive monocyte-to-macrophage differentiation through a mechanoepigenetic pathway. In vivo, differentiating monocytes exhibited flattened nuclei in the liver capsule, indicative of confinement by surrounding stromal and parenchymal structures. Using a custom cell confiner to recapitulate this confined niche, we found that confinement induces macrophage-like protrusive architectures, enhances motility, and upregulates macrophage-associated genes in RAW264.7 and THP-1 monocyte-lineage cells. Notably, extending this paradigm to primary murine bone-marrow, human umbilical-cord, and tissue-derived hepatic-associated monocytes yielded similar outcomes, thus enhancing phagocytic capacity, directly demonstrating that mechanical confinement can program monocytes into macrophages. Mechanistically, we found that confinement activates KDM6B, leading to H3K27me3 demethylation, which derepresses macrophage-specific transcriptional programs. Pharmacological inhibition of KDM6B with GSK-J4 restored H3K27me3 and blocked macrophage differentiation both in vitro and in vivo. These findings define a KDM6B-H3K27me3 axis that links nuclear mechanics to transcriptional reprogramming, positioning mechanical confinement as a "super-enhancer-like" cue for engineer macrophage function in therapeutic and bioengineering contexts. ### Competing Interest Statement The authors have declared no competing interest. National Natural Science Foundation of China, No. 22274026, No. 32401200 China Postdoctoral Science Foundation, No. 2024M760695 Postdoctoral Fellowship Program of CPSF, No. GZC20240297
Epidermal Growth factor (EGF) signaling is associated with (oncogenic) proliferation. Conversely, EGF-family ligands are able to trigger a differentiation program in cultured cells, an effect attributed to ligand affinity and EGFR phosphorylation. How EGF/EGFR driven proliferation-differentiation dynamics underlie tissue self-renewal has not been addressed. We show that culturing mouse small intestinal organoids (mSIOs) without EGF enhanced EGFR expression and base phosphorylation while maintaining a balanced development of proliferative crypts and differentiated villi. Addition of EGF or EREG triggers receptor endocytosis, reducing cell-surface and expression levels. While EGF promoted crypt proliferation, EREG promoted both proliferation and villus differentiation compared to untreated controls. Removal or re-introduction of EGF or EREG proved sufficient to induce development comparable to constant presence of ligands over 96h. Sub-saturating concentrations of EGF led to increased villus differentiation, resembling EREG treatments, suggesting that control over EGFR endocytic cycle ultimately regulates the balance of proliferation and differentiation in mSIOs ### Competing Interest Statement The authors have declared no competing interest.
Notch-mediated lateral inhibition is a conserved patterning process that controls alternative cell fate decisions and produces regular cell fate patterns. Prevailing models posit that lateral inhibition singles-out cells from fields of initially equipotent cells by amplifying stochastic fluctuations of Notch or pre-existing fate biases. Here, we revisited the role of Notch in early Drosophila neurogenesis, studying the dynamics of Neuroblast specification by live imaging the transcription of two proneural genes, scute and lethal of scute. We found that proneural gene expression is biased spatially along the dorsal-ventral axis prior to germ band extension and that early proneural expression predicts Neuroblast fate acquisition. This indicated that Neuroblast specification is pre-patterned by positional cues. Additionally, positional cues appeared to instruct individual cells to delaminate in a correct stereotyped pattern in proneural mutant embryos. Finally, contrary to current models, Notch signaling, measured by E(spl)m8 expression, was not detectable within proneural clusters until after Neuroblasts had initiated delamination. This indicated that Notch functions to stabilize rather than initiate fate decisions. We therefore propose that positional cues, not Notch, single-out Neuroblasts during early Drosophila neurogenesis, challenging long-held assumptions about the role of Notch in Neuroblast selection. ### Competing Interest Statement The authors have declared no competing interest. Agence Nationale de la Recherche, ANR-10-LABX-0073 Fondation pour la Recherche Médicale, FRM-DEQ20180339219
Quantifying chromatin-state dynamics in living cells remains challenging, in part because most methods require fixation or cell lysis. Here, we benchmark and introduce three simple live-cell image-derived metrics computed from routine DNA staining - the coefficient of variation (CV), 1-Gini, and the Diffuse Signal Index (DSI), introduced here - as fixation-free readouts of chromatin state. Using HL60-derived neutrophils (dHL-60) undergoing NETosis as a model system with a pronounced compact-to-decompact chromatin transition, we show that all three metrics track progressive chromatin reorganization in live-cell trajectories, but differ markedly in sensitivity: DSI provides the strongest trajectory-level discrimination between NETing and non-NETing cells, followed by 1-Gini and CV. Comparison with Tn5-based chromatin accessibility measurements in fixed cells further shows that all three metrics correlate with chromatin accessibility, supporting their biological relevance. Together, our results provide a practical framework for extracting chromatin-state readouts from routine live-cell DNA staining and identify DSI as the most discriminative metric for tracking chromatin reorganization in this benchmark. ### Competing Interest Statement The authors have declared no competing interest. Chan Zuckerberg Biohub San Francisco, https://ror.org/00knt4f32 David and Lucile Packard Foundation, https://ror.org/032atxq54
https://exquisite.tube/w/f3j7EAZQupCdj46EjWNPyJ
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Medusozoan cnidarians (e.g., jellyfish) metamorphose from a benthic juvenile polyp into a pelagic adult medusa, providing a well-known example of a clade that uses tissue remodeling to create distinct juvenile and adult body plans. Staurozoans (i.e., stalked jellyfish) are an atypical lineage of medusozoans that have lost their medusa stage; thus, their juvenile and adult body plans look remarkably alike. Their limited metamorphosis is characterized by the regression of primary (juvenile) tentacles and the development of secondary (adult) tentacles. In some staurozoan lineages, metamorphosis also involves development of novel adhesive structures (anchors), which are built on top of the regressing primary tentacles. Understanding how cells are partitioned from making juvenile tissues to making adult tissues is important for understanding how animals can make adult structures in the absence of complete metamorphosis. We compared the abundance and distribution of proliferative cells in tissues undergoing regression (primary tentacles) and development (secondary tentacles and anchors) during the juvenile to adult transition in the San Juan Island stalked jellyfish, Haliclystus sanjuanensis . We show that proliferative cells are lost in regressing primary tentacles but are gained in anchors, consistent with a shift in investment from juvenile to adult tissue. Prior to regression, primary and secondary tentacles show similar patterns in their proliferative cell distribution and in the identity of their cnidocytes (stinging cells), indicating that adult tentacles are made by re-deploying a juvenile tentacle program. Finally, we demonstrate that unlike secondary tentacles, primary tentacles cannot regenerate, illustrating that the temporary investment in this tissue is tied to their loss of proliferative cells. Thus, we propose that continued investment in a population of proliferating cells is an important mechanism for segregating temporary tissues (primary tentacles) from long-term tissues (secondary tentacles). These observations of cell dynamics in H. sanjuanensis suggest that temporary investment into juvenile structures may be used to pattern novel adult tissues, providing an important mechanism for diversifying adult body plans. ### Competing Interest Statement The authors have declared no competing interest. National Institutes of Health, NIGMS R35GM147253-01
In tunicates, larvaceans represent a fascinating case of evolution where the chordate body plan has been maintained despite a rapidly evolving genome characterized by strong compaction, massive chromosome rearrangements, gene losses and gene duplications. In contrast to other tunicates, larvaceans keep the chordate body plan during their entire life. They have acquired a highly specialized epithelium in charge of producing the »house», a complex extracellular apparatus used for filter feeding in the plankton. To what extent the house and this epithelium represent true molecular innovations withing chordates is a question for which thorough transcriptomics can bring novel insights. We conducted a developmental profiling of gene expression at the single-cell level in the larvacean Oikopleura dioica . We provide detailed descriptions of cellular transcriptomes associated with the house-synthesizing organ, which permits to define the molecular specifics of epithelial cell territories. We followed their emergence during development, and we identified genes that represent key candidate molecules for regulating the morphogenesis of the house-producing organ. Dynamic changes in gene expression and cell identities during major developmental transitions of the lifecycle illustrate that our dataset effectively allows access to the diversity of cell types in O. dioica embryos and in adults. The resources presented here constitute critical assets to investigate larvacean biology and evolution for mechanistic and comparative goals. ### Competing Interest Statement The authors have declared no competing interest. Research Council of Norway, 234817, 250005
Precise control over protein secretion is essential for programming intercellular communication and coordinating complex physiological responses. However, conventional methods relying on transcriptional regulation or chemical induction often lack the spatiotemporal precision and reversibility required to mimic endogenous signaling dynamics. Here, we present the Blue Light-Assisted Secretion Toolkit (BLAST), a genetically encoded system that orchestrates protein release from the endoplasmic reticulum via light-tunable protein-protein interactions. BLAST comprises two complementary modules utilizing both light-induced iLID/SspB association (a-BLAST) and LOV2/Zdk1 dissociation (d-BLAST). Both modules harness the highly conserved RXR motif to enforce strict ER confinement in the dark state. Most importantly, by utilizing non-destructive steric masking rather than enzymatic cleavage, BLAST achieves unprecedented temporal resolution with strict reversibility. We demonstrate that both systems can be repeatedly toggled ON and OFF, instantaneously arresting cargo release upon light withdrawal to generate highly controlled, pulsatile secretion profiles. Leveraging this dynamic control, we successfully achieved the rapid, robust, and light-triggered secretion of complex therapeutic proteins, including insulin and interleukin-12. By bypassing transcriptional delays and irreversible activation steps, BLAST provides a generalized, plug-and-play platform for the on-demand delivery of therapeutic proteins, significantly expanding the optogenetic toolbox for synthetic biology and cell-based therapies. ### Competing Interest Statement The authors have declared no competing interest. National Research Foundation of Korea, https://ror.org/013aysd81, RS-2020-NR051270, RS-2024-00340694
Godunov-type methods, which obtain numerical fluxes through local Riemann problems at cell interfaces, are among the most fundamental and widely used numerical methods in computational fluid dynamics. Exact Riemann solvers faithfully solve the underlying equations, but can be computationally expensive due to the iterative root-finding procedures they often require. Consequently, most practical computations rely on classical approximate Riemann solvers, such as Rusanov and Roe, which trade accuracy for computational speed. Neural networks have recently shown promise as an alternative for approximating exact Riemann solvers, but most existing approaches are data-driven or impose weak constraints. This may result in problems with maintaining balanced states, symmetry breaking, and conservation errors when integrated into a Godunov-type scheme. To address these issues, we propose a hard-constrained neural Riemann solver (HCNRS) and enforce five constraints: positivity, consistency, mirror symmetry, Galilean invariance, and scaling invariance. Numerical experiments are carried out for the shallow water and ideal-gas Euler equations on standard benchmark problems. In the absence of hard constraints, violations of the well-balanced property, mass conservation, and symmetry are observed. Notably, in the Euler implosion problem, the exact Riemann solver with MUSCL-Hancock captures the jet structure well, whereas the Rusanov flux is too diffusive and smears it out. HCNRS accurately reproduces the solution obtained by the exact Riemann solver. In contrast, an unconstrained neural formulation lacks mirror symmetry, which makes the solution depend on the choice of flux normal direction. As a result, the jet is either shifted or lost, along with diagonal symmetry.
Rxiv mechanobio
Senne Driesen