Peptide REF1 is a local wound signal promoting plant regeneration.

Plants frequently encounter wounding and have evolved an extraordinary regenerative capacity to heal the wounds. However, the wound signal that triggers regenerative responses has not been identified. Here, through characterization of a tomato mutant defective in both wound-induced defense and regeneration, we demonstrate that in tomato, a plant elicitor peptide (Pep), REGENERATION FACTOR1 (REF1), acts as a systemin-independent local wound signal that primarily regulates local defense responses and regenerative responses in response to wounding. We further identified PEPR1/2 ORTHOLOG RECEPTOR-LIKE KINASE1 (PORK1) as the receptor perceiving REF1 signal for plant regeneration. REF1-PORK1-mediated signaling promotes regeneration via activating WOUND-INDUCED DEDIFFERENTIATION 1 (WIND1), a master regulator of wound-induced cellular reprogramming in plants. Thus, REF1-PORK1 signaling represents a conserved phytocytokine pathway to initiate, amplify, and stabilize a signaling cascade that orchestrates wound-triggered organ regeneration. Application of REF1 provides a simple method to boost the regeneration and transformation efficiency of recalcitrant crops.

Transgenerational inheritance of acquired epigenetic signatures at CpG islands in mice.

Transgenerational epigenetic inheritance in mammals remains a debated subject. Here, we demonstrate that DNA methylation of promoter-associated CpG islands (CGIs) can be transmitted from parents to their offspring in mice. We generated DNA methylation-edited mouse embryonic stem cells (ESCs), in which CGIs of two metabolism-related genes, the Ankyrin repeat domain 26 and the low-density lipoprotein receptor, were specifically methylated and silenced. DNA methylation-edited mice generated by microinjection of the methylated ESCs exhibited abnormal metabolic phenotypes. Acquired methylation of the targeted CGI and the phenotypic traits were maintained and transmitted across multiple generations. The heritable CGI methylation was subjected to reprogramming in parental PGCs and subsequently reestablished in the next generation at post-implantation stages. These observations provide a concrete step toward demonstrating transgenerational epigenetic inheritance in mammals, which may have implications in our understanding of evolutionary biology as well as the etiology, diagnosis, and prevention of non-genetically inherited human diseases.

Ex utero monkey embryogenesis from blastocyst to early organogenesis.

The third and fourth weeks of gestation in primates are marked by several developmental milestones, including gastrulation and the formation of organ primordia. However, our understanding of this period is limited due to restricted access to in vivo embryos. To address this gap, we developed an embedded 3D culture system that allows for the extended ex utero culture of cynomolgus monkey embryos for up to 25 days post-fertilization. Morphological, histological, and single-cell RNA-sequencing analyses demonstrate that ex utero cultured monkey embryos largely recapitulated key events of in vivo development. With this platform, we were able to delineate lineage trajectories and genetic programs involved in neural induction, lateral plate mesoderm differentiation, yolk sac hematopoiesis, primitive gut, and primordial germ-cell-like cell development in monkeys. Our embedded 3D culture system provides a robust and reproducible platform for growing monkey embryos from blastocysts to early organogenesis and studying primate embryogenesis ex utero.

Hindbrain Double-Negative Feedback Mediates Palatability-Guided Food and Water Consumption.

Hunger and thirst have distinct goals but control similar ingestive behaviors, and little is known about neural processes that are shared between these behavioral states. We identify glutamatergic neurons in the peri-locus coeruleus (periLCVGLUT2 neurons) as a polysynaptic convergence node from separate energy-sensitive and hydration-sensitive cell populations. We develop methods for stable hindbrain calcium imaging in free-moving mice, which show that periLCVGLUT2 neurons are tuned to ingestive behaviors and respond similarly to food or water consumption. PeriLCVGLUT2 neurons are scalably inhibited by palatability and homeostatic need during consumption. Inhibition of periLCVGLUT2 neurons is rewarding and increases consumption by enhancing palatability and prolonging ingestion duration. These properties comprise a double-negative feedback relationship that sustains food or water consumption without affecting food- or water-seeking. PeriLCVGLUT2 neurons are a hub between hunger and thirst that specifically controls motivation for food and water ingestion, which is a factor that contributes to hedonic overeating and obesity.

Single-Cell Transcriptomic Atlas of Primate Ovarian Aging.

Molecular mechanisms of ovarian aging and female age-related fertility decline remain unclear. We surveyed the single-cell transcriptomic landscape of ovaries from young and aged non-human primates (NHPs) and identified seven ovarian cell types with distinct gene-expression signatures, including oocyte and six types of ovarian somatic cells. In-depth dissection of gene-expression dynamics of oocytes revealed four subtypes at sequential and stepwise developmental stages. Further analysis of cell-type-specific aging-associated transcriptional changes uncovered the disturbance of antioxidant signaling specific to early-stage oocytes and granulosa cells, indicative of oxidative damage as a crucial factor in ovarian functional decline with age. Additionally, inactivated antioxidative pathways, increased reactive oxygen species, and apoptosis were observed in granulosa cells from aged women. This study provides a comprehensive understanding of the cell-type-specific mechanisms underlying primate ovarian aging at single-cell resolution, revealing new diagnostic biomarkers and potential therapeutic targets for age-related human ovarian disorders.

Generation of Blastocyst-like Structures from Mouse Embryonic and Adult Cell Cultures.

A single mouse blastomere from an embryo until the 8-cell stage can generate an entire blastocyst. Whether laboratory-cultured cells retain a similar generative capacity remains unknown. Starting from a single stem cell type, extended pluripotent stem (EPS) cells, we established a 3D differentiation system that enabled the generation of blastocyst-like structures (EPS-blastoids) through lineage segregation and self-organization. EPS-blastoids resembled blastocysts in morphology and cell-lineage allocation and recapitulated key morphogenetic events during preimplantation and early postimplantation development in vitro. Upon transfer, some EPS-blastoids underwent implantation, induced decidualization, and generated live, albeit disorganized, tissues in utero. Single-cell and bulk RNA-sequencing analysis revealed that EPS-blastoids contained all three blastocyst cell lineages and shared transcriptional similarity with natural blastocysts. We also provide proof of concept that EPS-blastoids can be generated from adult cells via cellular reprogramming. EPS-blastoids provide a unique platform for studying early embryogenesis and pave the way to creating viable synthetic embryos by using cultured cells.

N6-methyladenine DNA Modification in Glioblastoma.

Genetic drivers of cancer can be dysregulated through epigenetic modifications of DNA. Although the critical role of DNA 5-methylcytosine (5mC) in the regulation of transcription is recognized, the functions of other non-canonical DNA modifications remain obscure. Here, we report the identification of novel N6-methyladenine (N6-mA) DNA modifications in human tissues and implicate this epigenetic mark in human disease, specifically the highly malignant brain cancer glioblastoma. Glioblastoma markedly upregulated N6-mA levels, which co-localized with heterochromatic histone modifications, predominantly H3K9me3. N6-mA levels were dynamically regulated by the DNA demethylase ALKBH1, depletion of which led to transcriptional silencing of oncogenic pathways through decreasing chromatin accessibility. Targeting the N6-mA regulator ALKBH1 in patient-derived human glioblastoma models inhibited tumor cell proliferation and extended the survival of tumor-bearing mice, supporting this novel DNA modification as a potential therapeutic target for glioblastoma. Collectively, our results uncover a novel epigenetic node in cancer through the DNA modification N6-mA.

m6Am sequesters PCF11 to suppress premature termination and drive neuroblastoma differentiation.

N6,2'-O-dimethyladenosine (m6Am) is an abundant mRNA modification that impacts multiple diseases, but its function remains controversial because the m6Am reader is unknown. Using quantitative proteomics, we identified transcriptional terminator premature cleavage factor II (PCF11) as a m6Am-specific reader in human cells. Direct quantification of mature versus nascent RNAs reveals that m6Am does not regulate mRNA stability but promotes nascent transcription. Mechanistically, m6Am functions by sequestering PCF11 away from proximal RNA polymerase II (RNA Pol II). This suppresses PCF11 from dissociating RNA Pol II near transcription start sites, thereby promoting full-length transcription of m6Am-modified RNAs. m6Am's unique relationship with PCF11 means m6Am function is enhanced when PCF11 is reduced, which occurs during all-trans-retinoic-acid (ATRA)-induced neuroblastoma-differentiation therapy. Here, m6Am promotes expression of ATF3, which represses neuroblastoma biomarker MYCN. Depleting m6Am suppresses MYCN repression in ATRA-treated neuroblastoma and maintains their tumor-stem-like properties. Collectively, we characterize m6Am as an anti-terminator RNA modification that suppresses premature termination and modulates neuroblastoma's therapeutic response.

RNA Pol II preferentially regulates ribosomal protein expression by trapping disassociated subunits.

RNA polymerase II (RNA Pol II) has been recognized as a passively regulated multi-subunit holoenzyme. However, the extent to which RNA Pol II subunits might be important beyond the RNA Pol II complex remains unclear. Here, fractions containing disassociated RPB3 (dRPB3) were identified by size exclusion chromatography in various cells. Through a unique strategy, i.e., "specific degradation of disassociated subunits (SDDS)," we demonstrated that dRPB3 functions as a regulatory component of RNA Pol II to enable the preferential control of 3' end processing of ribosomal protein genes directly through its N-terminal domain. Machine learning analysis of large-scale genomic features revealed that the little elongation complex (LEC) helps to specialize the functions of dRPB3. Mechanistically, dRPB3 facilitates CBC-PCF11 axis activity to increase the efficiency of 3' end processing. Furthermore, RPB3 is dynamically regulated during development and diseases. These findings suggest that RNA Pol II gains specific regulatory functions by trapping disassociated subunits in mammalian cells.

Injectable ultrasonic sensor for wireless monitoring of intracranial signals.

Direct and precise monitoring of intracranial physiology holds immense importance in delineating injuries, prognostication and averting disease1. Wired clinical instruments that use percutaneous leads are accurate but are susceptible to infection, patient mobility constraints and potential surgical complications during removal2. Wireless implantable devices provide greater operational freedom but include issues such as limited detection range, poor degradation and difficulty in size reduction in the human body3. Here we present an injectable, bioresorbable and wireless metastructured hydrogel (metagel) sensor for ultrasonic monitoring of intracranial signals. The metagel sensors are cubes 2 × 2 × 2 mm3 in size that encompass both biodegradable and stimulus-responsive hydrogels and periodically aligned air columns with a specific acoustic reflection spectrum. Implanted into intracranial space with a puncture needle, the metagel deforms in response to physiological environmental changes, causing peak frequency shifts of reflected ultrasound waves that can be wirelessly measured by an external ultrasound probe. The metagel sensor can independently detect intracranial pressure, temperature, pH and flow rate, realize a detection depth of 10 cm and almost fully degrade within 18 weeks. Animal experiments on rats and pigs indicate promising multiparametric sensing performances on a par with conventional non-resorbable wired clinical benchmarks.

Observation and quantification of the pseudogap in unitary Fermi gases.

The microscopic origin of high-temperature superconductivity in cuprates remains unknown. It is widely believed that substantial progress could be achieved by better understanding of the pseudogap phase, a normal non-superconducting state of cuprates1,2. In particular, a central issue is whether the pseudogap could originate from strong pairing fluctuations3. Unitary Fermi gases4,5, in which the pseudogap-if it exists-necessarily arises from many-body pairing, offer ideal quantum simulators to address this question. Here we report the observation of a pair-fluctuation-driven pseudogap in homogeneous unitary Fermi gases of lithium-6 atoms, by precisely measuring the fermion spectral function through momentum-resolved microwave spectroscopy and without spurious effects from final-state interactions. The temperature dependence of the pairing gap, inverse pair lifetime and single-particle scattering rate are quantitatively determined by analysing the spectra. We find a large pseudogap above the superfluid transition temperature. The inverse pair lifetime exhibits a thermally activated exponential behaviour, uncovering the microscopic virtual pair breaking and recombination mechanism. The obtained large, temperature-independent single-particle scattering rate is comparable with that set by the Planckian limit6. Our findings quantitatively characterize the pseudogap in strongly interacting Fermi gases and they lend support for the role of preformed pairing as a precursor to superfluidity.

Bound star clusters observed in a lensed galaxy 460 Myr after the Big Bang.

The Cosmic Gems arc is among the brightest and highly magnified galaxies observed at redshift z ≈ 10.2 (ref. 1). However, it is an intrinsically ultraviolet faint galaxy, in the range of those now thought to drive the reionization of the Universe2-4. Hitherto the smallest features resolved in a galaxy at a comparable redshift are between a few hundreds and a few tens of parsecs (pc)5,6. Here we report JWST observations of the Cosmic Gems. The light of the galaxy is resolved into five star clusters located in a region smaller than 70 pc. They exhibit minimal dust attenuation and low metallicity, ages younger than 50 Myr and intrinsic masses of about 106M⊙. Their lensing-corrected sizes are approximately 1 pc, resulting in stellar surface densities near 105M⊙ pc-2, three orders of magnitude higher than typical young star clusters in the local Universe7. Despite the uncertainties inherent to the lensing model, they are consistent with being gravitationally bound stellar systems, that is, proto-globular clusters. We conclude that star cluster formation and feedback likely contributed to shaping the properties of galaxies during the epoch of reionization.

Distinct structural bases for sequence-specific DNA binding by mammalian BEN domain proteins.

The BEN domain is a recently recognized DNA binding module that is present in diverse metazoans and certain viruses. Several BEN domain factors are known as transcriptional repressors, but, overall, relatively little is known of how BEN factors identify their targets in humans. In particular, X-ray structures of BEN domain:DNA complexes are only known for Drosophila factors bearing a single BEN domain, which lack direct vertebrate orthologs. Here, we characterize several mammalian BEN domain (BD) factors, including from two NACC family BTB-BEN proteins and from BEND3, which has four BDs. In vitro selection data revealed sequence-specific binding activities of isolated BEN domains from all of these factors. We conducted detailed functional, genomic, and structural studies of BEND3. We show that BD4 is a major determinant for in vivo association and repression of endogenous BEND3 targets. We obtained a high-resolution structure of BEND3-BD4 bound to its preferred binding site, which reveals how BEND3 identifies cognate DNA targets and shows differences with one of its non-DNA-binding BEN domains (BD1). Finally, comparison with our previous invertebrate BEN structures, along with additional structural predictions using AlphaFold2 and RoseTTAFold, reveal distinct strategies for target DNA recognition by different types of BEN domain proteins. Together, these studies expand the DNA recognition activities of BEN factors and provide structural insights into sequence-specific DNA binding by mammalian BEN proteins.

Olig2 targets chromatin remodelers to enhancers to initiate oligodendrocyte differentiation.

Establishment of oligodendrocyte identity is crucial for subsequent events of myelination in the CNS. Here, we demonstrate that activation of ATP-dependent SWI/SNF chromatin-remodeling enzyme Smarca4/Brg1 at the differentiation onset is necessary and sufficient to initiate and promote oligodendrocyte lineage progression and maturation. Genome-wide multistage studies by ChIP-seq reveal that oligodendrocyte-lineage determination factor Olig2 functions as a prepatterning factor to direct Smarca4/Brg1 to oligodendrocyte-specific enhancers. Recruitment of Smarca4/Brg1 to distinct subsets of myelination regulatory genes is developmentally regulated. Functional analyses of Smarca4/Brg1 and Olig2 co-occupancy relative to chromatin epigenetic marking uncover stage-specific cis-regulatory elements that predict sets of transcriptional regulators controlling oligodendrocyte differentiation. Together, our results demonstrate that regulation of the functional specificity and activity of a Smarca4/Brg1-dependent chromatin-remodeling complex by Olig2, coupled with transcriptionally linked chromatin modifications, is critical to precisely initiate and establish the transcriptional program that promotes oligodendrocyte differentiation and subsequent myelination of the CNS.

Large-scale across species transcriptomic analysis identifies genetic selection signatures associated with longevity in mammals.

Lifespan varies significantly among mammals, with more than 100-fold difference between the shortest and longest living species. This natural difference may uncover the evolutionary forces and molecular features that define longevity. To understand the relationship between gene expression variation and longevity, we conducted a comparative transcriptomics analysis of liver, kidney, and brain tissues of 103 mammalian species. We found that few genes exhibit common expression patterns with longevity in the three organs analyzed. However, pathways related to translation fidelity, such as nonsense-mediated decay and eukaryotic translation elongation, correlated with longevity across mammals. Analyses of selection pressure found that selection intensity related to the direction of longevity-correlated genes is inconsistent across organs. Furthermore, expression of methionine restriction-related genes correlated with longevity and was under strong selection in long-lived mammals, suggesting that a common strategy is utilized by natural selection and artificial intervention to control lifespan. Our results indicate that lifespan regulation via gene expression is driven through polygenic and indirect natural selection.

A transthyretin-like protein acts downstream of miR397 and LACCASE to regulate grain yield in rice.

Increasing grain yield is a major goal of breeders due to the rising global demand for food. We previously reported that the miR397-LACCASE (OsLAC) module regulates brassinosteroid (BR) signaling and grain yield in rice (Oryza sativa). However, the precise roles of laccase enzymes in the BR pathway remain unclear. Here, we report that OsLAC controls grain yield by preventing the turnover of TRANSTHYRETIN-LIKE (OsTTL), a negative regulator of BR signaling. Overexpressing OsTTL decreased BR sensitivity in rice, while loss-of-function of OsTTL led to enhanced BR signaling and increased grain yield. OsLAC directly binds to OsTTL and regulates its phosphorylation-mediated turnover. The phosphorylation site Ser226 of OsTTL is essential for its ubiquitination and degradation. Overexpressing the dephosphorylation-mimic form of OsTTL (OsTTLS226A) resulted in more severe defects than did overexpressing OsTTL. These findings provide insight into the role of an ancient laccase in BR signaling and suggest that the OsLAC-OsTTL module could serve as a target for improving grain yield.

Rad52 Restrains Resection at DNA Double-Strand Break Ends in Yeast.

Rad52 is a key factor for homologous recombination (HR) in yeast. Rad52 helps assemble Rad51-ssDNA nucleoprotein filaments that catalyze DNA strand exchange, and it mediates single-strand DNA annealing. We find that Rad52 has an even earlier function in HR in restricting DNA double-stranded break ends resection that generates 3' single-stranded DNA (ssDNA) tails. In fission yeast, Exo1 is the primary resection nuclease, with the helicase Rqh1 playing a minor role. We demonstrate that the choice of two extensive resection pathways is regulated by Rad52. In rad52 cells, the resection rate increases from ∼3-5 kb/h up to ∼10-20 kb/h in an Rqh1-dependent manner, while Exo1 becomes dispensable. Budding yeast Rad52 similarly inhibits Sgs1-dependent resection. Single-molecule analysis with purified budding yeast proteins shows that Rad52 competes with Sgs1 for DNA end binding and inhibits Sgs1 translocation along DNA. These results identify a role for Rad52 in limiting ssDNA generated by end resection.

WAV E3 ubiquitin ligases mediate degradation of IAA32/34 in the TMK1-mediated auxin signaling pathway during apical hook development.

Auxin regulates plant growth and development through downstream signaling pathways, including the best-known SCFTIR1/AFB-Aux/IAA-ARF pathway and several other less characterized "noncanonical" pathways. Recently, one SCFTIR1/AFB-independent noncanonical pathway, mediated by Transmembrane Kinase 1 (TMK1), was discovered through the analyses of its functions in Arabidopsis apical hook development. Asymmetric accumulation of auxin on the concave side of the apical hook triggers DAR1-catalyzed release of the C-terminal of TMK1, which migrates into the nucleus, where it phosphorylates and stabilizes IAA32/34 to inhibit cell elongation, which is essential for full apical hook formation. However, the molecular factors mediating IAA32/34 degradation have not been identified. Here, we show that proteins in the CYTOKININ INDUCED ROOT WAVING 1 (CKRW1)/WAVY GROWTH 3 (WAV3) subfamily act as E3 ubiquitin ligases to target IAA32/34 for ubiquitination and degradation, which is inhibited by TMK1c-mediated phosphorylation. This antagonistic interaction between TMK1c and CKRW1/WAV3 subfamily E3 ubiquitin ligases regulates IAA32/34 levels to control differential cell elongation along opposite sides of the apical hook.

Tbl1 promotes Wnt-ß-catenin signaling-induced degradation of the Tcf7l1 protein in mouse embryonic stem cells.

Activation of the Wnt-ß-catenin signaling pathway by CHIR99021, a specific inhibitor of GSK3ß, induces Tcf7l1 protein degradation, which facilitates the maintenance of an undifferentiated state in mouse embryonic stem cells (mESCs); however, the precise mechanism is still unclear. Here, we showed that the overexpression of transducin-ß-like protein 1 (Tbl1, also known as Tbl1x) or its family member Tblr1 (also known as Tbl1xr1) can decrease Tcf7l1 protein levels, whereas knockdown of each gene increases Tcf7l1 levels without affecting Tcf7l1 transcription. Interestingly, only Tbl1, and not Tblr1, interacts with Tcf7l1. Mechanistically, Tbl1 translocates from the cytoplasm into the nucleus in association with ß-catenin (CTNNB1) after the addition of CHIR99021 and functions as an adaptor to promote ubiquitylation of the Tcf7l1 protein. Functional assays further revealed that enforced expression of Tbl1 is capable of delaying mESC differentiation. In contrast, knockdown of Tbl1 attenuates the effect of CHIR99021 on Tcf7l1 protein stability and mESC self-renewal. Our results provide insight into the regulatory network of the Wnt-ß-catenin signaling pathway involved in promoting the maintenance of naïve pluripotency.

Global and local functions of the Fused kinase ortholog CdaH in intracellular patterning in Tetrahymena.

Ciliates assemble numerous microtubular structures into complex cortical patterns. During ciliate division, the pattern is duplicated by intracellular segmentation that produces a tandem of daughter cells. In Tetrahymena thermophila, the induction and positioning of the division boundary involves two mutually antagonistic factors: posterior CdaA (cyclin E) and anterior CdaI (Hippo kinase). Here, we characterized the related cdaH-1 allele, which confers a pleiotropic patterning phenotype including an absence of the division boundary and an anterior-posterior mispositioning of the new oral apparatus. CdaH is a Fused or Stk36 kinase ortholog that localizes to multiple sites that correlate with the effects of its loss, including the division boundary and the new oral apparatus. CdaH acts downstream of CdaA to induce the division boundary and drives asymmetric cytokinesis at the tip of the posterior daughter. CdaH both maintains the anterior-posterior position of the new oral apparatus and interacts with CdaI to pattern ciliary rows within the oral apparatus. Thus, CdaH acts at multiple scales, from induction and positioning of structures on the cell-wide polarity axis to local organelle-level patterning.