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5-Methylcytosine (5mC) is an established epigenetic mark in vertebrate genomic DNA, but whether its oxidation intermediates formed during TET-mediated DNA demethylation possess an instructive role of their own that is also physiologically relevant remains unresolved. Here, we reveal a 5-formylcytosine (5fC) nuclear chromocenter, which transiently forms during zygotic genome activation (ZGA) in Xenopus and mouse embryos. We identify this chromocenter as the perinucleolar compartment, a structure associated with RNA Pol III transcription. In Xenopus embryos, 5fC is highly enriched on Pol III target genes activated at ZGA, notably at oocyte-type tandem arrayed tRNA genes. By manipulating Tet and Tdg enzymes, we show that 5fC is required as a regulatory mark to promote Pol III recruitment as well as tRNA expression. Concordantly, 5fC modification of a tRNA transgene enhances its expression in vivo. The results establish 5fC as an activating epigenetic mark during zygotic reprogramming of Pol III gene expression. [Display omitted] • 5fC forms chromocenters in the perinucleolar compartment during Xenopus ZGA • 5fC colocalizes with RNA Pol III and accumulates on active tRNA tandem repeats • 5fC promotes Pol III recruitment and tRNA transcription at ZGA • 5fC stimulates transcription of a 5fC-modified tRNA transgene An open question in epigenetics is if 5-formylcytosine (5fC) is merely a byproduct of active demethylation or an instructive mark that regulates gene expression on its own. This work demonstrates 5fC chromocenters in early frog embryos and shows that 5fC acts as an instructive mark to promote RNA Pol III transcription during zygotic genome activation. [ABSTRACT FROM AUTHOR]

Collapse of membrane lipid asymmetry is a hallmark of blood coagulation. TMEM16F of the TMEM16 family that includes TMEM16A/B Ca(2+)-activated Cl(-) channels (CaCCs) is linked to Scott syndrome with deficient Ca(2+)-dependent lipid scrambling. We generated TMEM16F knockout mice that exhibit bleeding defects and protection in an arterial thrombosis model associated with platelet deficiency in Ca(2+)-dependent phosphatidylserine exposure and procoagulant activity and lack a Ca(2+)-activated cation current in the platelet precursor megakaryocytes. Heterologous expression of TMEM16F generates a small-conductance Ca(2+)-activated nonselective cation (SCAN) current with subpicosiemens single-channel conductance rather than a CaCC. TMEM16F-SCAN channels permeate both monovalent and divalent cations, including Ca(2+), and exhibit synergistic gating by Ca(2+) and voltage. We further pinpointed a residue in the putative pore region important for the cation versus anion selectivity of TMEM16F-SCAN and TMEM16A-CaCC channels. This study thus identifies a Ca(2+)-activated channel permeable to Ca(2+) and critical for Ca(2+)-dependent scramblase activity during blood coagulation. PAPERFLICK:

Summary Reproduction is heavily influenced by nutrition and metabolic state. Many common reproductive disorders in humans are associated with diabetes and metabolic syndrome. We characterized the metabolic mechanisms that support oogenesis and found that mitochondria in mature Drosophila oocytes enter a low-activity state of respiratory quiescence by remodeling the electron transport chain (ETC). This shift in mitochondrial function leads to extensive glycogen accumulation late in oogenesis and is required for the developmental competence of the oocyte. Decreased insulin signaling initiates ETC remodeling and mitochondrial respiratory quiescence through glycogen synthase kinase 3 (GSK3). Intriguingly, we observed similar ETC remodeling and glycogen uptake in maturing Xenopus oocytes, suggesting that these processes are evolutionarily conserved aspects of oocyte development. Our studies reveal an important link between metabolism and oocyte maturation. [ABSTRACT FROM AUTHOR]

Summary: Ten-Eleven Translocation (Tet) family of dioxygenases dynamically regulates DNA methylation and has been implicated in cell lineage differentiation and oncogenesis. Yet their functions and mechanisms of action in gene regulation and embryonic development are largely unknown. Here, we report that Xenopus Tet3 plays an essential role in early eye and neural development by directly regulating a set of key developmental genes. Tet3 is an active 5mC hydroxylase regulating the 5mC/5hmC status at target gene promoters. Biochemical and structural studies further demonstrate that the Tet3 CXXC domain is critical for specific Tet3 targeting. Finally, we show that the enzymatic activity and CXXC domain are both crucial for Tet3’s biological function. Together, these findings define Tet3 as a transcription regulator and reveal a molecular mechanism by which the 5mC hydroxylase and DNA binding activities of Tet3 cooperate to control target gene expression and embryonic development. [ABSTRACT FROM AUTHOR]

Summary: Secreted Wnt morphogens are signaling molecules essential for embryogenesis, pathogenesis, and regeneration and require distinct modifications for secretion, gradient formation, and activity. Whether Wnt proteins can be posttranslationally inactivated during development and homeostasis is unknown. Here we identify, through functional cDNA screening, a transmembrane protein Tiki1 that is expressed specifically in the dorsal Spemann-Mangold Organizer and is required for anterior development during Xenopus embryogenesis. Tiki1 antagonizes Wnt function in embryos and human cells via a TIKI homology domain that is conserved from bacteria to mammals and acts likely as a protease to cleave eight amino-terminal residues of a Wnt protein, resulting in oxidized Wnt oligomers that exhibit normal secretion but minimized receptor-binding capability. Our findings identify a Wnt-specific protease that controls head formation, reveal a mechanism for morphogen inactivation through proteolysis-induced oxidation-oligomerization, and suggest a role of the Wnt amino terminus in evasion of oxidizing inactivation. TIKI proteins may represent potential therapeutic targets. [ABSTRACT FROM AUTHOR]

Summary: Local protein synthesis plays a key role in regulating stimulus-induced responses in dendrites and axons. Recent genome-wide studies have revealed that thousands of different transcripts reside in these distal neuronal compartments, but identifying those with functionally significant roles presents a challenge. We performed an unbiased screen to look for stimulus-induced, protein synthesis-dependent changes in the proteome of Xenopus retinal ganglion cell (RGC) axons. The intermediate filament protein lamin B2 (LB2), normally associated with the nuclear membrane, was identified as an unexpected major target. Axonal ribosome immunoprecipitation confirmed translation of lb2 mRNA in vivo. Inhibition of lb2 mRNA translation in axons in vivo does not affect guidance but causes axonal degeneration. Axonal LB2 associates with mitochondria, and LB2-deficient axons exhibit mitochondrial dysfunction and defects in axonal transport. Our results thus suggest that axonally synthesized lamin B plays a crucial role in axon maintenance by promoting mitochondrial function. [Copyright &y& Elsevier]

Summary: Bipolar spindles must separate chromosomes by the appropriate distance during cell division, but mechanisms determining spindle length are poorly understood. Based on a 2D model of meiotic spindle assembly, we predicted that higher localized microtubule (MT) depolymerization rates could generate the shorter spindles observed in egg extracts of X. tropicalis compared to X. laevis. We found that katanin-dependent MT severing was increased in X. tropicalis, which, unlike X. laevis, lacks an inhibitory phosphorylation site in the katanin p60 catalytic subunit. Katanin inhibition lengthened spindles in both species. In X. tropicalis, k-fiber MT bundles that connect to chromosomes at their kinetochores extended through spindle poles, disrupting them. In both X. tropicalis extracts and the spindle simulation, a balance between k-fiber number and MT depolymerization is required to maintain spindle morphology. Thus, mechanisms have evolved in different species to scale spindle size and coordinate regulation of multiple MT populations in order to generate a robust steady-state structure. [Copyright &y& Elsevier]

Summary: The eukaryotic replicative DNA helicase, CMG, unwinds DNA by an unknown mechanism. In some models, CMG encircles and translocates along one strand of DNA while excluding the other strand. In others, CMG encircles and translocates along duplex DNA. To distinguish between these models, replisomes were confronted with strand-specific DNA roadblocks in Xenopus egg extracts. An ssDNA translocase should stall at an obstruction on the translocation strand but not the excluded strand, whereas a dsDNA translocase should stall at obstructions on either strand. We found that replisomes bypass large roadblocks on the lagging strand template much more readily than on the leading strand template. Our results indicate that CMG is a 3′ to 5′ ssDNA translocase, consistent with unwinding via “steric exclusion.” Given that MCM2-7 encircles dsDNA in G1, the data imply that formation of CMG in S phase involves remodeling of MCM2-7 from a dsDNA to a ssDNA binding mode. [Copyright &y& Elsevier]

Summary: DNA interstrand crosslinks (ICLs) are toxic DNA lesions whose repair occurs in the S phase of metazoans via an unknown mechanism. Here, we describe a cell-free system based on Xenopus egg extracts that supports ICL repair. During DNA replication of a plasmid containing a site-specific ICL, two replication forks converge on the crosslink. Subsequent lesion bypass involves advance of a nascent leading strand to within one nucleotide of the ICL, followed by incisions, translesion DNA synthesis, and extension of the nascent strand beyond the lesion. Immunodepletion experiments suggest that extension requires DNA polymerase ζ. Ultimately, a significant portion of the input DNA is fully repaired, but not if DNA replication is blocked. Our experiments establish a mechanism for ICL repair that reveals how this process is coupled to DNA replication. [Copyright &y& Elsevier]

Summary: For chromosomes to congress and segregate during cell division, kinetochores must form stable attachments with spindle microtubules. We find that the centrosome protein, xCep57, localizes to kinetochores and interacts with the kinetochore proteins Zwint, Mis12, and CLIP-170. Immunodepletion of xCep57 from egg extracts yields weakened and elongated bipolar spindles which fail to align chromosomes. In the absence of xCep57, tension is lost between sister kinetochores, and spindle microtubules are no longer resistant to low doses of nocodazole. xCep57 inhibition on isolated mitotic chromosomes inhibits kinetochore-microtubule binding in vitro. xCep57 also interacts with γ-tubulin. In xCep57 immunodepleted extracts, sperm centrosomes nucleate with normal kinetics, but are unable maintain microtubule anchorage. This characterization places xCep57 in a novel class of proteins required for stable microtubule attachments at the kinetochore and at the centrosome and suggests that the mechanism of microtubule binding at these two places is mechanistically similar. [Copyright &y& Elsevier]

Summary: A novel model for the regulation of cell excitability has recently been proposed. It originates from the observation that the background K+ channel K2P1 (TWIK1) may be silenced by sumoylation in Xenopus oocytes and that inactivation of the putative sumoylation site (mutation K274E) gives rise to robust current expression in transfected COS-7 cells. Here, we show that only the mutation K274E, and not K274R, is associated with an increase of K2P1 current density, suggesting a charge effect of K274E. Furthermore, we failed to observe any band shift by western blot analysis that would confirm an eventual sumoylation of K2P1 in COS-7 cells and oocytes. [Copyright &y& Elsevier]

Summary: Here we report an unexpected role for the secreted Frizzled-related protein (sFRP) Sizzled/Ogon as an inhibitor of the extracellular proteolytic reaction that controls BMP signaling during Xenopus gastrulation. Microinjection experiments suggest that the Frizzled domain of Sizzled regulates the activity of Xolloid-related (Xlr), a metalloproteinase that degrades Chordin, through the following molecular pathway: Szl ┤ Xlr ┤ Chd ┤ BMP → P-Smad1 → Szl. In biochemical assays, the Xlr proteinase has similar affinities for its endogenous substrate Chordin and for its competitive inhibitor Sizzled, which is resistant to enzyme digestion. Extracellular levels of Sizzled and Chordin in the gastrula embryo and enzyme reaction constants were all in the 10−8 M range, consistent with a physiological role in the regulation of dorsal-ventral patterning. Sizzled is also a natural inhibitor of BMP1, a Tolloid metalloproteinase of medical interest. Furthermore, mouse sFRP2 inhibited Xlr, suggesting a wider role for this molecular mechanism. [Copyright &y& Elsevier]

Summary: Embryos have the ability to self-regulate and regenerate normal structures after being sectioned in half. How is such a morphogenetic field established? We discovered that quadruple knockdown of ADMP and BMP2/4/7 in Xenopus embryos eliminates self-regulation, causing ubiquitous neural induction throughout the ectoderm. ADMP transcription in the Spemann organizer is activated at low BMP levels. When ventral BMP2/4/7 signals are depleted, Admp expression increases, allowing for self-regulation. ADMP has BMP-like activity and signals via the ALK-2 receptor. It is unable to signal dorsally because of inhibition by Chordin. The ventral BMP antagonists Sizzled and Bambi further refine the pattern. By transplanting dorsal or ventral wild-type grafts into ADMP/BMP2/4/7-depleted hosts, we demonstrate that both poles serve as signaling centers that can induce histotypic differentiation over considerable distances. We conclude that dorsal and ventral BMP signals and their extracellular antagonists expressed under opposing transcriptional regulation provide a molecular mechanism for embryonic self-regulation. [Copyright &y& Elsevier]

Wnt signaling pathways play essential roles in patterning and proliferation of embryonic and adult tissues. In many organisms, this signaling pathway directs axis formation. Although the importance of intracellular components of the pathway, including β-catenin and Tcf3, has been established, the mechanism of their activation is uncertain. In Xenopus, the initiating signal that localizes β-catenin to dorsal nuclei has been suggested to be intracellular and Wnt independent. Here, we provide three lines of evidence that the pathway specifying the dorsal axis is activated extracellularly in Xenopus embryos. First, we identify Wnt11 as the initiating signal. Second, we show that activation requires the glycosyl transferase X.EXT1. Third, we find that the EGF-CFC protein, FRL1, is also essential and interacts with Wnt11 to activate canonical Wnt signaling. [ABSTRACT FROM AUTHOR]

Investigates the distinct origins of adult and embryonic blood in Xenopus. Derivation of embryonic and adult blood from a single or dual origin; First adult hematopoietic stem cells; Aortic clusters in Xenopus; Aortic clusters derived from the blastomere; Origins of blood in vertebrate embryos.

Develops a method to purify metaphase and anaphase chromosomes from frog egg extracts to identify proteins that leave chromosomes at anaphase using a new form of expression screening. Background on process of mitosis; Role of Xenopus homolog Xkid in localizing metaphase and chromosome alignment; Strategy for identifying chromosomal proteins.

Examines the induction of Muller glia from retinoblasts in Xenopus by p27Xic1. Analysis of gliogenic function of p27Xic1; Order of laminar differentiation in retina and cortex; Determination of glial cells in Xenopus retina; Role of cyclin-dependent kinase inhibitors in genetic determination.

Examines role of SNAP-25 in the activation of the store-operated Ca2+ current in Xenopus oocytes. Mechanism of Ca2+ influx; Inhibition of Rho guanosine triphosphatase; Role of botulinum neurotoxins (BoNT) in ISOC activation; Comparisons of the effects of brefeldin A , BoNT A and SNAP-25; Reexamination of the diffusable messenger hypothesis.

Focuses on a study which evaluated the effects of overexpression of XOptx2 in the Xenopus embryonic eyefield. Information on the isolation and molecular analysis of Xenopus laevis; Effect of XOptx2 to vertebrate retinal development.

Presents information on a study which showed that maternally expressed VegT controls is the pattern of primary germ layer specification in Xenopus embryos. Overview of the study; Results; Discussion.

Provides information on a study which identifies the role of glutamine binding proteins (GBP), a maternal Xgsk-3-binding proteins, on the establishment of the dorsal-ventral axis in Xenopus. Cloning and expression of a novel Xgsk-3-binding proteins; Effect of GBP on axis duplication; Role of GBP on the regulation of stable beta-catenin.

Focuses on a study in which two Xenopus relatives of murine Sox17 were isolated and expressed in gastrula presumptive endoderm. Induction of the Xenopus relatives in animal caps by activin; Information on the Ectopic expression of the genes; Blocking the induction; Discussion of the results of the study.

Characterizes X-MyT1, a Xenopus C2HC-type zinc finger protein. Involvement of X-MyT1 in the primary selection of neuronal precursor cells; Regulation of X-Myt1 gene expression by the bHLH protein X-NGNR-1 and Notch/Delta signal transduction pathway; Promotion of ectopic neuronal differentiation.

Describes the role of Eomesodermin or Eomes, a T-domain gene, in Xenopus mesoderm differentiation. Eomesodermin expression in a ventral-to-dorsal gradient; Time for peak expression; Effect of ectopic expression; Overexpression result on ventral mesoderm and cell fate; Outcomes of function blockage.

Studies the interaction between the origin recognition complex and the replication licensing system in Xenopus. Involvement of XORC in DNA replication; Chromatin association of XOrc1; Amino acid sequence of the XOrc1 protein; Effect of immunodepletion of XOrc1 on DNA replication; Sequential assemblies.

Characterizes the role of the Cdc6 homolog protein in DNA replication of Xenopus egg extracts. Abolition of the chromosomal replication by immunodepleted Xcdc6 homolog; Chromatin binding of the protein; Requirement of the protein component called Xorc2 for the homolog's binding; Interaction with nuclear envelop regulating the homolog's functions.

Examines the dorsoventral patterning in xenopus. Inhibition of ventral signals by the direct binding of chordin to BMP-4; Secretion of chordin through the Spemann organizer tissue during gastrulation Effects of the dorsal tissue grafted to the ventral side; Ectodermal patterning.

Describes the XTcf-3 transcription factor that mediates beta-catenin-induced axis formation in Xenophus embryos. Identification of four Tcf/Lef homologs; Translocation beta-catenin to the nucleus; Suppression of axis formation by deltaN mutant of XTcf-3.

Looks at the results of the cloning of Xenopus cDNAs homologous to the Drosophila Mad gene, and C. elegans CEM genes, which were analyzed by microinjection into frog embryos. Analysis of the results; What the results suggest.

Describes a novel Xenopus plus end-directed kinesin-like protein (KLP), Xklp2, localized on centrosomes throughout the cell cycle and on spindle pole microtubules during metaphase. Use of mitotic spindles assembled in Xenopus egg extracts; Evidence that Xklp2 protein is required for centrosome separation.

Assesses the mechanisms whereby Xenopus nodal-related 3 (Xnr3) may contribute to tissue differentiation of the embryo. Xnr3 as found only in the epithelial component of the Spemann organizer; Xnr3 attributes; Possible role of Xnr3 in regulating differentiation and morphogenesis in the gastrula embryo; Xnr3 as the possible functional equivalent of the mouse nodal.

Reports on the identification of the yeast MCM3-related protein as a component of Xenopus DNA replication licensing factor. Behavior during cell cycle; Results of cloning and sequencing of the cDNAs; Transport of Xenopus p100 into the nuclei.

Examines the effects of injection of wild-type and mutant SH2-containing protein-tyrosine phosphatase SH-PTP2 RNAs on Xenopus embryogenesis. Internal phosphatase domain deletion as a dominant negative mutant of SH-PTP2; Blocking of fibroblast growth factor- and activin-mediated mesoderm induction; Requirement of SH-PTP2 upstream of MAP kinase.

Assesses the role of cytoplasmic polyadenylation element-binding protein (CPEB) in cytoplasmic polyadenylation. Isolation of the CPEB by affinity chromatography; Developmental expression of CPEB; Phosphorylation of CPEB during Xenopus oocyte maturation.

Identifies ERK2, a member of the mitogen-activated protein (MAP) kinase family as a component of the spindle assembly checkpoint in Xenopus. Stabilization of the cyclin B by spindle assembly checkpoint; Lesions that activate the checkpoint; Role of calcium in overcoming M phase arrests.

Describes an isolation procedure for mitotic chromosomes assembled in Xenopus egg extracts which allowed the identification of chromosomal peptides, XCAP-C and XCAP-E. XCAP-C/E structures; Characterization of protein components of mitotic chromosomes assembled in vitro; Localization of XCAP-C/E; Roles of XCAP-C/E in structural maintenance of mitotic chromosome.

Reports that nuclear events typical of apoptosis can be reproduced by Xenopus egg extracts. Initial normality of nuclear assembly and protein import; Process of nuclear destruction; Bcl-2 as a block to its effect.

Studies the role of Xenopus bone morphogenic protein (BMP) receptor in mesoderm induction in embryos. Expression of the BMP receptor during embryonic development; Relationship between induction in the ventral and dorsal mesoderm.

Discusses a role for transcription and FRGY2 in masking maternal mRNA within Xenopus oocytes. Mechanisms of masking mRNA from translation; Multifuncional RNA-binding proteins and the transcription process; Experimental procedures.

Studies the regulation of gene expression during early Xenopus development due to competition between chromatin and transcription complex assembly. Role of TATA binding protein (TBP) in the repression of transcription; Necessity of potentiating templates with TBP for premature transcription; Role of chromatin assembly in repressing transcription.

Studies the role of inhibition of activin receptor signaling in neuralization of Xenopus. Expression of a truncated activin type II receptor; Identification of molecules involve in formation of vertebrate nervous system; Diversion of prospective ectodermal and endodermal blastomeres to a neural fate.

Presents a molecular characterization of the swelling-induced chloride conductance regulatory protein plcln. Identification and localization; Cloning in Xenopus oocytes; Formation of multiple oligomeric complexes with cytosolic proteins.

Studies the role of Vg1 protein in the Xenopus blastula stage. TGF-beta-related growth factor; Encoding by maternal messenger ribonucleic acid (mRNA); Concentration in vegetal blastomeres; Generation of dorsal mesoderm and the body axis in Xenopus.

Investigates the role of calcium during nuclear envelope assembly in vitro using Xenopus egg extracts. Effect of 5mM BAPTA; ADP-ribosylation factor; Exogenous inositol 1, 4, 5-triphosphate (IP3); Role of heparin; Benefit of phosphoinositide signaling.

Nucleotide-binding, leucine-rich repeat receptors (NLRs) are major immune receptors in plants and animals. Upon activation, the Arabidopsis NLR protein ZAR1 forms a pentameric resistosome in vitro and triggers immune responses and cell death in plants. In this study, we employed single-molecule imaging to show that the activated ZAR1 protein can form pentameric complexes in the plasma membrane. The ZAR1 resistosome displayed ion channel activity in Xenopus oocytes in a manner dependent on a conserved acidic residue Glu11 situated in the channel pore. Pre-assembled ZAR1 resistosome was readily incorporated into planar lipid-bilayers and displayed calcium-permeable cation-selective channel activity. Furthermore, we show that activation of ZAR1 in the plant cell led to Glu11-dependent Ca 2+ influx, perturbation of subcellular structures, production of reactive oxygen species, and cell death. The results thus support that the ZAR1 resistosome acts as a calcium-permeable cation channel to trigger immunity and cell death., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)