METTL3 regulates cartilage development and homeostasis by affecting Lats1 mRNA stability in an m6A-YTHDF2-dependent manner.

Cartilage maintains the structure and function of joints, with disturbances leading to potential osteoarthritis. N6-methyladenosine (m6A), the most widespread post-transcriptional modification in eukaryotes, plays a crucial role in regulating biological processes. While current research has indicated that m6A affects the progression of osteoarthritis, its function in the development and homeostasis of articular cartilage remains unclear. Here we report that Mettl3 deficiency in chondrocytes leads to mandibular condylar cartilage morphological alterations, early temporomandibular joint osteoarthritis, and diminished adaptive response to abnormal mechanical stimuli. Mechanistically, METTL3 modulates Lats1 mRNA methylation and facilitates its degradation in an m6A-YTHDF2-dependent manner, which subsequently influences the degradation and nuclear translocation of YAP1. Intervention with the Hippo pathway inhibitor XMU-MP-1 alleviates condylar abnormality caused by Mettl3 knockout. Our findings demonstrate the role of METTL3 in cartilage development and homeostasis, offering insights into potential treatment strategies for osteoarthritis.

The lateral habenula integrates age and experience to promote social transitions in developing rats.

Early caregiving adversity (ECA) is associated with social behavior deficits and later development of psychopathology. However, the infant neural substrates of ECA are poorly understood. The lateral habenula (LHb), a highly conserved brain region with consistent links to adult psychopathology, is understudied in development, when the brain is most vulnerable to environmental impacts. Here, we describe the structural and functional ontogeny of the LHb and its behavioral role in infant and juvenile rat pups. We show that the LHb promotes a developmental transition in social approach behavior under threat as typically reared infants mature. By contrast, we show that ECA disrupts habenular ontogeny, including volume, protein expression, firing properties, and corticohabenular connectivity. Furthermore, inhibiting a specific corticohabenular projection rescues infant social approach deficits following ECA. Together, these results identify immediate biomarkers of ECA in the LHb and highlight this region as a site of early social processing and behavior control.

Activated dormant stem cells recover spermatogenesis in chemoradiotherapy-induced infertility.

Male infertility is a recognized side effect of chemoradiotherapy. Extant spermatogonial stem cells (SSCs) may act as originators for any subsequent recovery. However, which type of SSCs, the mechanism by which they survive and resist toxicity, and how they act to restart spermatogenesis remain largely unknown. Here, we identify a small population of Set domain-containing protein 4 (Setd4)-expressing SSCs that occur in a relatively dormant state in the mouse seminiferous tubule. Extant beyond high-dose chemoradiotherapy, these cells then activate to recover spermatogenesis. Recovery fails when Setd4+ SSCs are deleted. Confirmed to be of fetal origin, these Setd4+ SSCs are shown to facilitate early testicular development and also contribute to steady-state spermatogenesis in adulthood. Upon activation, chromatin remodeling increases their genome-wide accessibility, enabling Notch1 and Aurora activation with corresponding silencing of p21 and p53. Here, Setd4+ SSCs are presented as the originators of both testicular development and spermatogenesis recovery in chemoradiotherapy-induced infertility.

A hominoid-specific signaling axis regulating the tempo of synaptic maturation.

Human cortical neurons (hCNs) exhibit high dendritic complexity and synaptic density, and the maturation process is greatly protracted. However, the molecular mechanism governing these specific features remains unclear. Here, we report that the hominoid-specific gene TBC1D3 promotes dendritic arborization and protracts the pace of synaptogenesis. Ablation of TBC1D3 in induced hCNs causes reduction of dendritic growth and precocious synaptic maturation. Forced expression of TBC1D3 in the mouse cortex protracts synaptic maturation while increasing dendritic growth. Mechanistically, TBC1D3 functions via interaction with MICAL1, a monooxygenase that mediates oxidation of actin filament. At the early stage of differentiation, the TBC1D3/MICAL1 interaction in the cytosol promotes dendritic growth via F-actin oxidation and enhanced actin dynamics. At late stages, TBC1D3 escorts MICAL1 into the nucleus and downregulates the expression of genes related with synaptic maturation through interaction with the chromatin remodeling factor ATRX. Thus, this study delineates the molecular mechanisms underlying human neuron development.

Dynamic regulation of alternative polyadenylation by PQBP1 during neurogenesis.

Alternative polyadenylation (APA) is a critical post-transcriptional process that generates mRNA isoforms with distinct 3' untranslated regions (3' UTRs), thereby regulating mRNA localization, stability, and translational efficiency. Cell-type-specific APA extensively shapes the diversity of the cellular transcriptome, particularly during cell fate transition. Despite its recognized significance, the precise regulatory mechanisms governing cell-type-specific APA remain unclear. In this study, we uncover PQBP1 as an emerging APA regulator that actively maintains cell-specific APA profiles in neural progenitor cells (NPCs) and delicately manages the equilibrium between NPC proliferation and differentiation. Multi-omics analysis shows that PQBP1 directly interacts with the upstream UGUA elements, impeding the recruitment of the CFIm complex and influencing polyadenylation site selection within genes associated with the cell cycle. Our findings elucidate the molecular mechanism by which PQBP1 orchestrates dynamic APA changes during neurogenesis, providing valuable insights into the precise regulation of cell-type-specific APA and the underlying pathogenic mechanisms in neurodevelopmental disorders.

The architecture of silk-secreting organs during the final larval stage of silkworms revealed by single-nucleus and spatial transcriptomics.

Natural silks are renewable proteins with impressive mechanical properties and biocompatibility that are useful in various fields. However, the cellular and spatial organization of silk-secreting organs remains unclear. Here, we combined single-nucleus and spatially resolved transcriptomics to systematically map the cellular and spatial composition of the silk glands (SGs) of mulberry silkworms late in larval development. This approach allowed us to profile SG cell types and cell state dynamics and identify regulatory networks and cell-cell communication related to efficient silk protein synthesis; key markers were validated via transgenic approaches. Notably, we demonstrated the indispensable role of the ecdysone receptor (ultraspiracle) in regulating endoreplication in SG cells. Our atlas presents the results of spatiotemporal analysis of silk-secreting organ architecture late in larval development; this atlas provides a valuable reference for elucidating the mechanism of efficient silk protein synthesis and developing sustainable products made from natural silk.

DCX knockout ferret reveals a neurogenic mechanism in cortical development.

Lissencephaly is a rare brain malformation for which our understanding remains limited due to the absence of suitable animal models that accurately represent human phenotypes. Here, we establish doublecortin (DCX) knockout ferrets as a model that faithfully replicates key features of the disorder. We reveal the critical roles of DCX in neural progenitor cell proliferation and radial glial fiber extension, processes essential for normal cortical development. Utilizing single-nucleus RNA sequencing (snRNA-seq) and spatial transcriptomics, we provide a detailed atlas of the lissencephalic cortex, illustrating disrupted neuronal lamination and the specific interactions between inhibitory and excitatory neurons. These findings enhance our understanding of the cellular and molecular mechanisms underlying lissencephaly and highlight the potential of DCX knockout ferrets as a valuable tool for neurodevelopmental research, offering insights into both the pathology of lissencephaly and the general principles of brain development.

Fatecode enables cell fate regulator prediction using classification-supervised autoencoder perturbation.

Cell reprogramming, which guides the conversion between cell states, is a promising technology for tissue repair and regeneration, with the ultimate goal of accelerating recovery from diseases or injuries. To accomplish this, regulators must be identified and manipulated to control cell fate. We propose Fatecode, a computational method that predicts cell fate regulators based only on single-cell RNA sequencing (scRNA-seq) data. Fatecode learns a latent representation of the scRNA-seq data using a deep learning-based classification-supervised autoencoder and then performs in silico perturbation experiments on the latent representation to predict genes that, when perturbed, would alter the original cell type distribution to increase or decrease the population size of a cell type of interest. We assessed Fatecode's performance using simulations from a mechanistic gene-regulatory network model and scRNA-seq data mapping blood and brain development of different organisms. Our results suggest that Fatecode can detect known cell fate regulators from single-cell transcriptomics datasets.

Cell-size-dependent regulation of Ezrin dictates epithelial resilience to stretch by countering myosin-II-mediated contractility.

The epithelial adaptations to mechanical stress are facilitated by molecular and tissue-scale changes that include the strengthening of junctions, cytoskeletal reorganization, and cell-proliferation-mediated changes in tissue rheology. However, the role of cell size in controlling these properties remains underexplored. Our experiments in the zebrafish embryonic epidermis, guided by theoretical estimations, reveal a link between epithelial mechanics and cell size, demonstrating that an increase in cell size compromises the tissue fracture strength and compliance. We show that an increase in E-cadherin levels in the proliferation-deficient epidermis restores epidermal compliance but not the fracture strength, which is largely regulated by Ezrin-an apical membrane-cytoskeleton crosslinker. We show that Ezrin fortifies the epithelium in a cell-size-dependent manner by countering non-muscle myosin-II-mediated contractility. This work uncovers the importance of cell size maintenance in regulating the mechanical properties of the epithelium and fostering protection against future mechanical stresses.

Macrophages enhance contractile force in iPSC-derived human engineered cardiac tissue.

Resident cardiac macrophages are critical mediators of cardiac function. Despite their known importance to cardiac electrophysiology and tissue maintenance, there are currently no stem-cell-derived models of human engineered cardiac tissues (hECTs) that include resident macrophages. In this study, we made an induced pluripotent stem cell (iPSC)-derived hECT model with a resident population of macrophages (iM0) to better recapitulate the native myocardium and characterized their impact on tissue function. Macrophage retention within the hECTs was confirmed via immunofluorescence after 28 days of cultivation. The inclusion of iM0s significantly impacted hECT function, increasing contractile force production. A potential mechanism underlying these changes was revealed by the interrogation of calcium signaling, which demonstrated the modulation of ß-adrenergic signaling in +iM0 hECTs. Collectively, these findings demonstrate that macrophages significantly enhance cardiac function in iPSC-derived hECT models, emphasizing the need to further explore their contributions not only in healthy hECT models but also in the contexts of disease and injury.

Wg/Wnt-signaling-induced nuclear translocation of ß-catenin is attenuated by a ß-catenin peptide through its interference with the IFT-A complex.

Wnt/Wingless (Wg) signaling is critical in development and disease, including cancer. Canonical Wnt signaling is mediated by ß-catenin/Armadillo (Arm in Drosophila) transducing signals to the nucleus, with IFT-A/Kinesin 2 complexes promoting nuclear translocation of ß-catenin/Arm. Here, we demonstrate that a conserved small N-terminal Arm34-87/ß-catenin peptide binds to IFT140, acting as a dominant interference tool to attenuate Wg/Wnt signaling in vivo. Arm34-87 expression antagonizes endogenous Wnt/Wg signaling, resulting in the reduction of its target expression. Arm34-87 inhibits Wg/Wnt signaling by interfering with nuclear translocation of endogenous Arm/ß-catenin, and this can be modulated by levels of wild-type ß-catenin or IFT140, with the Arm34-87 effect being enhanced or suppressed. Importantly, this mechanism is conserved in mammals with the equivalent ß-catenin24-79 peptide blocking nuclear translocation and pathway activation, including in cancer cells. Our work indicates that Wnt signaling can be regulated by a defined N-terminal ß-catenin peptide and thus might serve as an entry point for therapeutic applications to attenuate Wnt/ß-catenin signaling.

A cis-regulatory module underlies retinal ganglion cell genesis and axonogenesis.

Atoh7 is transiently expressed in retinal progenitor cells (RPCs) and is required for retinal ganglion cell (RGC) differentiation. In humans, a deletion in a distal non-coding regulatory region upstream of ATOH7 is associated with optic nerve atrophy and blindness. Here, we functionally interrogate the significance of the Atoh7 regulatory landscape to retinogenesis in mice. Deletion of the Atoh7 enhancer structure leads to RGC deficiency, optic nerve hypoplasia, and retinal blood vascular abnormalities, phenocopying inactivation of Atoh7. Further, loss of the Atoh7 remote enhancer impacts ipsilaterally projecting RGCs and disrupts proper axonal projections to the visual thalamus. Deletion of the Atoh7 remote enhancer is also associated with the dysregulation of axonogenesis genes, including the derepression of the axon repulsive cue Robo3. Our data provide insights into how Atoh7 enhancer elements function to promote RGC development and optic nerve formation and highlight a key role of Atoh7 in the transcriptional control of axon guidance molecules.

Reproductive regulation of the mitochondrial stress response in Caenorhabditis elegans.

Proteome integrity is fundamental for cellular and organismal homeostasis. The mitochondrial unfolded protein response (UPRmt), a key component of the proteostasis network, is activated in a non-cell-autonomous manner in response to mitochondrial stress in distal tissues. However, the importance of inter-tissue communication for UPRmt inducibility under physiological conditions remains elusive. Here, we show that an intact germline is essential for robust UPRmt induction in the Caenorhabditis elegans somatic tissues. A series of nematode mutants with germline defects are unable to respond to genetic or chemical UPRmt inducers. Our genetic analysis suggests that reproductive signals, rather than germline stem cells, are responsible for somatic UPRmt induction. Consistent with this observation, we show that UPRmt is sexually dimorphic, as male nematodes are inherently unresponsive to mitochondrial stress. Our findings highlight a paradigm of germline-somatic communication and suggest that reproductive cessation is a primary cause of age-related UPRmt decline.

Defining the cellular origin of seminoma by transcriptional and epigenetic mapping to the normal human germline.

Aberrant male germline development can lead to the formation of seminoma, a testicular germ cell tumor. Seminomas are biologically similar to primordial germ cells (PGCs) and many bear an isochromosome 12p [i(12p)] with two additional copies of the short arm of chromosome 12. By mapping seminoma transcriptomes and open chromatin landscape onto a normal human male germline trajectory, we find that seminoma resembles premigratory/migratory PGCs; however, it exhibits enhanced germline and pluripotency programs and upregulation of genes involved in apoptosis, angiogenesis, and MAPK/ERK pathways. Using pluripotent stem cell-derived PGCs from Pallister-Killian syndrome patients mosaic for i(12p), we model seminoma and identify gene dosage effects that may contribute to transformation. As murine seminoma models do not exist, our analyses provide critical insights into genetic, cellular, and signaling programs driving seminoma transformation, and the in vitro platform developed herein permits evaluation of additional signals required for seminoma tumorigenesis.

Progenitor-like cells contributing to cellular heterogeneity in the nucleus pulposus are lost in intervertebral disc degeneration.

The nucleus pulposus (NP) in the intervertebral disc (IVD) arises from embryonic notochord. Loss of notochordal-like cells in humans correlates with onset of IVD degeneration, suggesting that they are critical for healthy NP homeostasis and function. Comparative transcriptomic analyses identified expression of progenitor-associated genes (GREM1, KRT18, and TAGLN) in the young mouse and non-degenerated human NP, with TAGLN expression reducing with aging. Lineage tracing using Tagln-CreERt2 mice identified peripherally located proliferative NP (PeriNP) cells in developing and postnatal NP that provide a continuous supply of cells to the entire NP. PeriNP cells were diminished in aged mice and absent in puncture-induced degenerated discs. Single-cell transcriptomes of postnatal Tagln-CreERt2 IVD cells indicate enrichment for TGF-ß signaling in Tagln descendant NP sub-populations. Notochord-specific removal of TGF-ß/BMP mediator Smad4 results in loss of Tagln+ cells and abnormal NP morphologies. We propose Tagln+ PeriNP cells are potential progenitors crucial for NP homeostasis.

Piezo regulates epithelial topology and promotes precision in organ size control.

Mechanosensitive Piezo channels regulate cell division, cell extrusion, and cell death. However, systems-level functions of Piezo in regulating organogenesis remain poorly understood. Here, we demonstrate that Piezo controls epithelial cell topology to ensure precise organ growth by integrating live-imaging experiments with pharmacological and genetic perturbations and computational modeling. Notably, the knockout or knockdown of Piezo increases bilateral asymmetry in wing size. Piezo's multifaceted functions can be deconstructed as either autonomous or non-autonomous based on a comparison between tissue-compartment-level perturbations or between genetic perturbation populations at the whole-tissue level. A computational model that posits cell proliferation and apoptosis regulation through modulation of the cutoff tension required for Piezo channel activation explains key cell and tissue phenotypes arising from perturbations of Piezo expression levels. Our findings demonstrate that Piezo promotes robustness in regulating epithelial topology and is necessary for precise organ size control.

Connexin hemichannels drive lactation-induced osteocyte acidification and perilacunar-canalicular remodeling.

The maternal skeleton experiences significant bone loss during lactation, followed by rapid restoration post weaning. Parathyroid-related protein (PTHrP)-induced acidification of the perilacunar matrix by osteocytes is crucial in this process, yet its mechanism remains unclear. Here, we identify Cx43 hemichannels (HCs) as key mediators of osteocyte acidification and perilacunar-canalicular remodeling (PLR). Utilizing transgenic mouse models expressing dominant-negative Cx43 mutants, we show that mice with impaired Cx43 HCs exhibit attenuated lactation-induced responses compared to wild-type and only gap junction-impaired groups, including lacunar enlargement, upregulation of PLR genes, and bone loss with compromised mechanical properties. Furthermore, inhibition of HCs by a Cx43 antibody blunts PTHrP-induced calcium influx and protein kinase A activation, followed by impaired osteocyte acidification. Additionally, impeded HCs suppress bone recovery during the post-lactation period. Our findings highlight the pivotal role of Cx43 HCs in orchestrating dynamic bone changes during lactation and recovery by regulating acidification and remodeling enzyme expression.

Hair follicles modulate skin barrier function.

Our skin provides a protective barrier that shields us from our environment. Barrier function is typically associated with the interfollicular epidermis; however, whether hair follicles influence this process remains unclear. Here, we utilize a potent genetic tool to probe barrier function by conditionally ablating a quintessential epidermal barrier gene, Abca12, which is mutated in the most severe skin barrier disease, harlequin ichthyosis. With this tool, we deduced 4 ways by which hair follicles modulate skin barrier function. First, the upper hair follicle (uHF) forms a functioning barrier. Second, barrier disruption in the uHF elicits non-cell-autonomous responses in the epidermis. Third, deleting Abca12 in the uHF impairs desquamation and blocks sebum release. Finally, barrier perturbation causes uHF cells to move into the epidermis. Neutralizing IL-17a, whose expression is enriched in the uHF, partially alleviated some disease phenotypes. Altogether, our findings implicate hair follicles as multi-faceted regulators of skin barrier function.

AdamTS proteases control basement membrane heterogeneity and organ shape in Drosophila.

The basement membrane (BM) is an extracellular matrix that plays important roles in animal development. A spatial heterogeneity in composition and structural properties of the BM provide cells with vital cues for morphogenetic processes such as cell migration or cell polarization. Here, using the Drosophila egg chamber as a model system, we show that the BM becomes heterogeneous during development, with a reduction in Collagen IV density at the posterior pole and differences in the micropattern of aligned fiber-like structures. We identified two AdamTS matrix proteases required for the proper elongated shape of the egg chamber, yet the molecular mechanisms by which they act are different. Stall is required to establish BM heterogeneity by locally limiting Collagen IV protein density, whereas AdamTS-A alters the micropattern of fiber-like structures within the BM at the posterior pole. Our results suggest that AdamTS proteases control BM heterogeneity required for organ shape.

Epidermal turnover in the planarian Schmidtea mediterranea involves basal cell extrusion and intestinal digestion.

Planarian flatworms undergo continuous internal turnover, wherein old cells are replaced by the division progeny of adult pluripotent stem cells (neoblasts). How cell turnover is carried out at the organismal level remains an intriguing question in planarians and other systems. While previous studies have predominantly focused on neoblast proliferation, little is known about the processes that mediate cell loss during tissue homeostasis. Here, we use the planarian epidermis as a model to study the mechanisms of cell removal. We established a covalent dye-labeling assay and image analysis pipeline to quantify the cell turnover rate in the planarian epidermis. Our findings indicate that the ventral epidermis is highly dynamic and epidermal cells undergo internalization via basal extrusion, followed by a relocation toward the intestine and ultimately digestion by intestinal phagocytes. Overall, our study reveals a complex homeostatic process of cell clearance that may generally allow planarians to catabolize their own cells.