Mammalian spermatogenesis is a highly stereotyped and conserved developmental process that is essential for fitness. At the same time, gene expression in spermatogenic cells is rapidly evolving. This combination of features has been suggested to drive rapid fixation of new gene expression patterns. Using a high-resolution dataset comprising bulk and single-cell data from juvenile and adult testes of the opossum Monodelphis domestica, a model marsupial, we define the developmental timing of the spermatogenic first wave in opossum and delineate conserved and divergent gene expression programs across the placental-marsupial split by comparison to equivalent data from mouse, a model placental mammal. Epigenomic data confirmed divergent regulation at the level of transcription, and comparison to data from four additional amniote species identified hundreds of genes with evidence of rapid fixation of expression. This gene set encompasses known and previously undescribed regulators of spermatogenic development.

Changes in light quality and quantity experienced by many shade-intolerant plants grown in close proximity lead to transcriptional reprogramming and shade avoidance syndrome (SAS). Despite the importance of phosphorylation-dependent signaling in cellular physiology, phosphorylation events during SAS are largely unknown. Here, we examined shade-regulated phosphorylation events in Arabidopsis using quantitative phosphoproteomics. We confirmed shade-induced dephosphorylation of bZIP59, a basic region/leucine zipper motif (bZIP) transcription factor. Shade treatment promotes the nuclear localization of bZIP59, which can be mimicked by mutation of the phosphorylation sites on bZIP59. Phenotypic analysis identified that bZIP59 negatively regulated shade-induced hypocotyl elongation. bZIP59 repressed the shade-induced activation of certain growth-related genes, while shade increased the DNA binding of bZIP59. Furthermore, the protein phosphatase 2A (PP2A) mediated dephosphorylation of bZIP59. Our study characterized a previously unidentified mechanism by which the phytochrome B (phyB)-PP2A-bZIP59 regulatory module integrates shade signals and transcriptomes, broadening our knowledge of phosphorylation strategies for rapid adaptation to shade.

Layer-specific brain computations depend on neurons synapsing with specific partners in distinct laminae. In the Drosophila lobula plate, axons of the four subtypes of T4 and T5 visual motion direction-selective neurons segregate into four layers, where they synapse with distinct subsets of postsynaptic neurons. Here, we identify a layer-specific expression of different receptor-ligand pairs of the Beat and Side families of cell adhesion molecules between T4/T5s and their postsynaptic partners. Developmental genetic analysis demonstrate that Beat/Side-mediated interactions are required to restrict innervation of T4/T5 axons and the dendrites of their partners to a single layer. We show that Beat/Side interactions are not required for synaptogenesis. Instead, they contribute to synaptic specificity by biasing cellular adjacency, causing neurons to segregate in discrete layers, restricting partner availability before synaptogenesis. We propose that the emergence of synaptic specificity relies on a competitive dynamic among postsynaptic partners with shared Beat/Side expression to adhere with T4/T5s.

Tunneling nanotubes (TNTs) are thin intercellular connections that facilitate the transport of diverse cargoes, ranging from ions to organelles. While TNT studies have predominantly been conducted in cell cultures, the existence of open-ended TNTs within live organisms remains unverified. Despite the observation of intercellular connections during embryonic development across various species, their functional role in facilitating material transfer between connected cells has not been confirmed. In this study, we performed mosaic labeling of gastrula cells in zebrafish embryos to demonstrate the coexistence of TNT-like structures alongside other cellular protrusions. These embryonic TNT-like connections exhibited a morphology similar to that of TNTs described in cell culture, appeared to have similar formation mechanisms, and could be induced by Eps8 overexpression and CK666 treatment. Most notably, we demonstrated their capability to transfer both soluble cargoes and organelles, thus confirming their open-endedness. This study demonstrates the existence of functional, open-ended TNTs in a living embryo.

The cellular proteome determines the functional state of cells and is often skewed to direct pathological conditions. Autophagy shapes cellular proteomes primarily through lysosomal degradation of either damaged or unnecessary proteins. Here, we show that autophagy directs the senescence-specific translatome to fuel inflammation by coupling selective protein degradation with alternative splicing. RNA splicing is significantly altered during senescence, some of which surprisingly depend on autophagy, including exon 5 skipping of the translation regulator EIF4H. Systematic translatome profiling indicates that this event is key to the translational bias toward inflammation in senescence. Autophagy promotes these changes by selectively degrading the splicing regulator splicing factor proline and glutamine rich (SFPQ) via the autophagy receptor NBR1. These autophagy-centric inflammatory controls appear to be conserved during human tissue aging and cancer. Our work highlights the role of autophagy in the on-demand functional remodeling of cellular proteomes as well as the crosstalk between autophagy, alternative splicing, and inflammatory translation.

Pulsatile activity of the extracellular signal-regulated kinase (ERK) controls several cellular, developmental, and regenerative programs. Sequential segmentation of somites along the vertebrate body axis, a key developmental program, is also controlled by ERK activity oscillation. The oscillatory expression of Her/Hes family transcription factors constitutes the segmentation clock, setting the period of segmentation. Although oscillation of ERK activity depends on Her/Hes proteins, the underlying molecular mechanism remained mysterious. Here, we show that Her/Hes proteins physically interact with and stabilize dual-specificity phosphatases (Dusp) of ERK, resulting in oscillations of Dusp4 and Dusp6 proteins. Pharmaceutical and genetic inhibition of Dusp activity disrupt ERK activity oscillation and somite segmentation in zebrafish. Our results demonstrate that post-translational interactions of Her/Hes transcription factors with Dusp phosphatases establish the fundamental vertebrate body plan. We anticipate that future studies will identify currently unnoticed post-translational control of ERK pulses in other systems.

The rapidly regenerating intestinal epithelium requires crypt intestinal stem cells (ISCs). Wnt/β-catenin signaling maintains crypt homeostasis and Lgr5+ ISCs, and WNT ligands bind Frizzled receptors (FZD1-10). Identifying specific FZD(s) essential for intestinal homeostasis has been elusive; however, bioengineered antagonists blocking Wnt binding to FZD5 and FZD8 deplete the gut epithelium in vivo, highlighting potential roles. Here, an epithelial-specific Fzd5 knockout (KO) elicited lethal pan-intestinal crypt and villus loss, whereas an Lgr5+ ISC-specific Fzd5 KO depleted Lgr5+ ISCs via premature differentiation and repressed Wnt target genes. Fzd5-null phenotypes were rescued by constitutive β-catenin activation in vivo and in both mouse and human enteroids. KO of Fzd5, not Fzd8, in enteroids ablated responsiveness to dual-specificity FZD5/FZD8-selective Wnt surrogate agonists, which ameliorated DSS-induced colitis in wild-type and Fzd8 KO mice. Overall, FZD5 is a dominant and essential regulator of crypt homeostasis, Lgr5+ ISCs, and intestinal response to Wnt surrogate agonists, with implications for therapeutic mucosal repair.

Neutrophils in the tumor microenvironment (TME) are heterogeneous populations associated with cancer prognosis and immunotherapy. However, the plasticity and function of heterogeneous neutrophils in the TME of non-small-cell lung cancer (NSCLC) remain unclear. Here, we show that neutrophils produce high levels of interleukin (IL)-8, which induce the differentiation of CD74SiglecF neutrophils and suppress the generation of CD74SiglecF neutrophils in the TME of IL-8-humanized NSCLC mice. The CD74SiglecF neutrophils boost anti-tumor T cell responses via antigen cross-presentation. Deleting CD74 in IL-8-humanized neutrophils impairs T cell activation and exacerbates NSCLC progression, whereas a CD74 agonist enhances T cell activation and the efficacy of anti-programmed cell death 1 (PD-1) or osimertinib therapies. Additionally, the CD74CD63 neutrophils in the TME and peripheral blood of advanced NSCLC patients phenocopy the CD74SiglecF neutrophils in the TME of NSCLC mice and correlate well with the responsiveness to anti-PD-1 plus chemotherapies. These findings demonstrate an IL-8-CD74 neutrophil axis that promotes anti-tumor immunity in NSCLC.

During differentiation, cells become structurally and functionally specialized, but comprehensive views of the underlying remodeling processes are elusive. Here, we leverage single-cell RNA sequencing (scRNA-seq) developmental trajectories to reconstruct differentiation using two secretory tissues as models-the zebrafish notochord and hatching gland. First, we integrated expression and functional similarities to identify gene modules, revealing dozens of modules representing known and newly associated differentiation processes and their dynamics. Second, we focused on the unfolded protein response (UPR) transducer module to study how general versus cell-type-specific secretory functions are regulated. Profiling loss- and gain-of-function embryos identified that the UPR transcription factors creb3l1, creb3l2, and xbp1 are master regulators of a general secretion program. creb3l1/creb3l2 additionally activate an extracellular matrix secretion program, while xbp1 partners with bhlha15 to activate a gland-like secretion program. Our study presents module identification via multi-source integration for reconstructing differentiation (MIMIR) and illustrates how transcription factors confer general and specialized cellular functions.

The homeostasis of the intestinal epithelium relies on intricate yet insufficiently understood mechanisms of intestinal epithelial plasticity. Here, we elucidate the pivotal role of Frizzled5 (Fzd5), a Wnt pathway receptor, as a determinant of murine intestinal epithelial cell fate. Deletion of Fzd5 in Lgr5 intestinal stem cells (ISCs) impairs their self-renewal, whereas its deletion in Krt19 cells disrupts lineage generation, without affecting crypt integrity in either case. However, a broader deletion of Fzd5 across the epithelium leads to substantial crypt deterioration. Integrated analysis of single-cell RNA sequencing (scRNA-seq) and single-cell ATAC-seq (scATAC-seq) identifies that Fzd5 governs chromatin accessibility, orchestrating the regulation of stem- and lineage-related gene expression mainly in ISCs and progenitor cells. In summary, our findings provide insights into the regulatory role of Fzd5 in governing intestinal epithelial plasticity.

Plants demonstrate a high degree of developmental plasticity, capable of regenerating entire individuals from detached somatic tissues-a regenerative phenomenon rarely observed in metazoa. Consequently, elucidating the lineage relationship between somatic founder cells and descendant cells in regenerated plant organs has long been a pursuit. In this study, we developed and optimized both DNA barcode- and multi-fluorescence-based cell-lineage tracing toolsets, employing an inducible method to mark individual cells in Arabidopsis donor somatic tissues at the onset of regeneration. Utilizing these complementary methods, we scrutinized cell identities at the single-cell level and presented compelling evidence that all cells in the regenerated Arabidopsis plants, irrespective of their organ types, originated from a single progenitor cell in the donor somatic tissue. Our discovery suggests a single-cell passage directing the transition from multicellular donor tissue to regenerated plants, thereby creating opportunities for cell-cell competition during plant regeneration-a strategy for maximizing survival.

Amphibians, by virtue of their phylogenetic position, provide invaluable insights on nervous system evolution, development, and remodeling. The genetic toolkit for amphibians, however, remains limited. Recombinant adeno-associated viral vectors (AAVs) are a powerful alternative to transgenesis for labeling and manipulating neurons. Although successful in mammals, AAVs have never been shown to transduce amphibian cells efficiently. We screened AAVs in three amphibian species-the frogs Xenopus laevis and Pelophylax bedriagae and the salamander Pleurodeles waltl-and identified at least two AAV serotypes per species that transduce neurons. In developing amphibians, AAVs labeled groups of neurons generated at the same time during development. In the mature brain, AAVrg retrogradely traced long-range projections. Our study introduces AAVs as a tool for amphibian research, establishes a generalizable workflow for AAV screening in new species, and expands opportunities for cross-species comparisons of nervous system development, function, and evolution.

Developing tissues interpret dynamic changes in morphogen activity to generate cell type diversity. To quantitatively study bone morphogenetic protein (BMP) signaling dynamics in the mouse neural tube, we developed an embryonic stem cell differentiation system tailored for growing tissues. Differentiating cells form striking self-organized patterns of dorsal neural tube cell types driven by sequential phases of BMP signaling that are observed both in vitro and in vivo. Data-driven biophysical modeling showed that these dynamics result from coupling fast negative feedback with slow positive regulation of signaling by the specification of an endogenous BMP source. Thus, in contrast to relays that propagate morphogen signaling in space, we identify a BMP signaling relay that operates in time. This mechanism allows for a rapid initial concentration-sensitive response that is robustly terminated, thereby regulating balanced sequential cell type generation. Our study provides an experimental and theoretical framework to understand how signaling dynamics are exploited in developing tissues.

The Drosophila adult midgut progenitor cells (AMPs) give rise to all cells in the adult midgut epithelium, including the intestinal stem cells (ISCs). While they share many characteristics with the ISCs, it remains unclear how they are generated in the early embryo. Here, we show that they arise from a population of endoderm cells, which exhibit multiple similarities with Drosophila neuroblasts. These cells, which we have termed endoblasts, are patterned by homothorax (Hth) and undergo asymmetric divisions using the same molecular machinery as neuroblasts. We also show that the conservation of this molecular machinery extends to the generation of the enteroendocrine lineages. Parallels have previously been drawn between the pupal ISCs and larval neuroblasts. Our results suggest that these commonalities exist from the earliest stages of specification of progenitor cells of the intestinal and nervous systems and may represent an ancestral pathway for multipotent progenitor cell specification.

The human blastocyst contains the pluripotent epiblast from which human embryonic stem cells (hESCs) can be derived. ACTIVIN/NODAL signaling maintains expression of the transcription factor NANOG and in vitro propagation of hESCs. It is unknown whether this reflects a functional requirement for epiblast development in human embryos. Here, we characterized NODAL signaling activity during pre-implantation human development. We showed that NANOG is an early molecular marker restricted to the nascent human pluripotent epiblast and was initiated prior to the onset of NODAL signaling. We further demonstrated that expression of pluripotency-associated transcription factors NANOG, SOX2, OCT4, and KLF17 were maintained in the epiblast in the absence of NODAL signaling activity. Genome-wide transcriptional analysis showed that NODAL signaling inhibition did not decrease NANOG transcription or impact the wider pluripotency-associated gene regulatory network. These data suggest differences in the signaling requirements regulating pluripotency in the pre-implantation human epiblast compared with existing hESC culture.

Long-range transcriptional activation of gene promoters by abundant enhancers in animal genomes calls for mechanisms to limit inappropriate regulation. DNA elements called insulators serve this purpose by shielding promoters from an enhancer when interposed. Unlike promoters and enhancers, insulators have not been systematically characterized due to lacking high-throughput screening assays, and questions regarding how insulators are distributed and encoded in the genome remain. Here, we establish "insulator-seq" as a plasmid-based massively parallel reporter assay in Drosophila cultured cells to perform a systematic insulator screen of selected genomic loci. Screening developmental gene loci showed that not all insulator protein binding sites effectively block enhancer-promoter communication. Deep insulator mutagenesis identified sequences flexibly positioned around the CTCF insulator protein binding motif that are critical for functionality. The ability to screen millions of DNA sequences without positional effect has enabled functional mapping of insulators and provided further insights into the determinants of insulators.

During mouse preimplantation development, a subset of retrotransposons/genes are transiently expressed in the totipotent 2-cell (2C) embryos. These 2C transcripts rapidly shut down their expression beyond the 2C stage of embryos, promoting the embryo to exit from the 2C stage. However, the mechanisms regulating this shutdown remain unclear. Here, we identified that lysosomal catabolism played a role in the exit of the totipotent 2C state. Our results showed that the activation of embryonic lysosomal catabolism promoted the embryo to exit from the 2C stage and suppressed 2C transcript expression. Mechanistically, our results indicated that lysosomal catabolism suppressed 2C transcripts through replenishing cellular amino-acid levels, thereby inactivating transcriptional factors TFE3/TFEB and abolishing their transcriptional activation of 2C retrotransposons, MERVL (murine endogenous retrovirus-L)/MT2_Mm. Collectively, our study identified that lysosomal activity modulated the transcriptomic landscape and development in mouse embryos and identified an unanticipated layer of transcriptional control on early-embryonic retrotransposons from lysosomal signaling.

The ability of cancer cells to undergo identity changes (i.e., lineage plasticity) plays a key role in tumor progression and response to therapy. Loss of the pulmonary lineage specifier NKX2-1 in KRAS-driven lung adenocarcinoma (LUAD) enhances tumor progression and causes a FoxA1/2-dependent pulmonary-to-gastric lineage switch. However, the mechanisms by which FoxA1/2 activate a latent gastric identity in the lung remain largely unknown. Here, we show that FoxA1/2 reprogram the epigenetic landscape of gastric-specific genes after NKX2-1 loss in mouse models by facilitating ten-eleven translocation (TET)2/3 recruitment, DNA demethylation, histone 3 lysine 27 acetylation (H3K27ac) deposition, and three-dimensional (3D) chromatin interactions. FoxA1/2-mediated DNA methylation changes are highly conserved in human endodermal development and in progression of human lung and pancreatic neoplasia. Furthermore, oncogenic signaling is required for specific elements of FoxA1/2-dependent epigenetic reprogramming. This work demonstrates the role of FoxA1/2 in rewiring the DNA methylation and 3D chromatin landscape of NKX2-1-negative LUAD to drive cancer cell lineage switching.

The coordinated spatial arrangement of organelles within a tissue plane, known as planar cell polarity (PCP), is critical for organ development and function. Gradients of morphogens and their receptors typically set-up PCP, but whether non-molecular cues, akin to phototropism in plants, also play a part remains unknown. Here, we report that basal bodies of newborn photoreceptor cells in the mouse retina are positioned centrally on the apical surface but then move laterally during the first postnatal week, generating cell-intrinsic asymmetry in the retinal plane. After 1 week, when the eyes open, basal bodies of cone cilia, but not rods, become coordinated across the plane to face the center of the retina. We further show that light is essential for cone PCP, triggering a cascade in which cone transducin interacts with the G-protein-signaling modulator protein 2 (GPSM2) to establish PCP. This work identifies a non-canonical PCP pathway initiated by light.

The relapse of tongue cancer (TC) after chemotherapy is caused by minimal residual disease (MRD), which is a few remaining cancer cells after chemotherapy. To understand the mechanism of MRD in TC, we created a library of TC organoids (TCOs) from 28 untreated TC patients at diverse ages and cancer stages. These TCOs reproduced the primary TC tissues both in vitro and in a xenograft model, and several TCO lines survived after cisplatin treatment (chemo-resistant TCOs). Of note, the chemo-resistant TCOs showed "heritable" embryonic diapause-like features before treatment and activation of the autophagy and cholesterol biosynthetic pathways. Importantly, inhibiting these pathways with specific inhibitors converted the chemo-resistant TCOs into chemo-sensitive TCOs. Conversely, autophagy activation with mTOR inhibitors conferred chemo-resistance on the chemo-sensitive TCOs. This unique model provides insights into the mechanism of MRD formation in TCs, leading to effective therapeutic approaches to reduce the recurrence of TC.

The complexity of regeneration extends beyond local wound responses, eliciting systemic processes across the entire organism. However, the functional relevance and coordination of distant molecular processes remain unclear. In the cnidarian Nematostella vectensis, we show that local regeneration triggers a systemic homeostatic response, leading to coordinated whole-body remodeling. Leveraging spatial transcriptomics, endogenous protein tagging, and live imaging, we comprehensively dissect this systemic response at the organismal scale. We identify proteolysis as a critical process driven by both local and systemic upregulation of metalloproteases. We show that metalloproteinase expression levels and activity scale with the extent of tissue loss. This proportional response drives long-range tissue and extracellular matrix movement. Our findings demonstrate the adaptive nature of the systematic response in regeneration, enabling the organism to maintain shape homeostasis while coping with a wide range of injuries.