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Apical cell-cell junctions, including adherens junctions and tight junctions, adhere epithelial cells to one another and regulate selective permeability at both bicellular junctions and tricellular junctions (TCJs). Although several specialized proteins are known to localize at TCJs, it remains unclear how actomyosin-mediated tension transmission at TCJs contributes to the maintenance of junction integrity and barrier function at these sites. Here, utilizing the embryonic epithelium of gastrula-stage Xenopus laevis embryos, we define a mechanism by which the mechanosensitive protein Vinculin helps anchor the actomyosin network at TCJs, thus maintaining TCJ integrity and barrier function. Using an optogenetic approach to acutely increase junctional tension, we find that Vinculin is mechanosensitively recruited to apical junctions immediately surrounding TCJs. In Vinculin knockdown (KD) embryos, junctional actomyosin intensity is decreased and becomes disorganized at TCJs. Using fluorescence recovery after photobleaching (FRAP), we show that Vinculin KD reduces actin stability at TCJs and destabilizes Angulin-1, a key tricellular tight junction protein involved in regulating barrier function at TCJs. When Vinculin KD embryos are subjected to increased tension, TCJ integrity is not maintained, filamentous actin (F-actin) morphology at TCJs is disrupted, and breaks in the signal of the tight junction protein ZO-1 signal are detected. Finally, using a live imaging barrier assay, we detect increased barrier leaks at TCJs in Vinculin KD embryos. Together, our findings show that Vinculin-mediated actomyosin organization is required to maintain junction integrity and barrier function at TCJs and reveal new information about the interplay between adhesion and barrier function at TCJs. [Display omitted] • Vinculin is mechanosensitively recruited to tricellular junctions • Vinculin's actin-binding function is needed for tricellular actomyosin organization • Vinculin reinforces tricellular junction integrity in response to increased tension • Vinculin is required to maintain barrier function at tricellular junctions Epithelial tricellular junctions are vulnerable to disruption. Here, van den Goor et al. reveal that Vinculin is mechanosensitively recruited to vertebrate tricellular junctions, where it regulates actomyosin organization and barrier function. Vinculin reinforces tricellular junction integrity when epithelia experience increased mechanical force. [ABSTRACT FROM AUTHOR]

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]

Deubiquitylating enzymes (DUBs) remove ubiquitin from proteins thereby regulating their stability or activity. Our understanding of DUB-substrate specificity is limited because DUBs are typically not compared to each other against many physiological substrates. By broadly inhibiting DUBs in Xenopus egg extract, we generated hundreds of ubiquitylated proteins and compared the ability of 30 DUBs to deubiquitylate them using quantitative proteomics. We identified five high-impact DUBs (USP7, USP9X, USP36, USP15, and USP24) that each reduced ubiquitylation of over 10% of the isolated proteins. Candidate substrates of high-impact DUBs showed substantial overlap and were enriched for disordered regions, suggesting this feature may promote substrate recognition. Other DUBs showed lower impact and non-overlapping specificity, targeting distinct non-disordered proteins including complexes such as the ribosome or the proteasome. Altogether our study identifies candidate DUB substrates and defines patterns of functional redundancy and specificity, revealing substrate characteristics that may influence DUB-substrate recognition. [Display omitted] • DUB profiling against endogenously ubiquitylated proteins reveals new substrates • DUBs vary widely in their ability to deubiquitylate different proteins • A subset of DUBs can deubiquitylate a large number of proteins • DUB substrates are more likely to be disordered than non-substrates Here, Rossio et al. develop a method to directly compare the activity of deubiquitylating enzymes (DUBs) against each other. This approach revealed hundreds of new candidate substrates, providing new insights into DUB specificity and function. The results suggest that substrate disorder may favor recognition by some DUBs. [ABSTRACT FROM AUTHOR]

Repair of DNA double-strand breaks by the non-homologous end-joining pathway is initiated by the binding of Ku to DNA ends. Multiple Ku proteins load onto linear DNAs in vitro. However, in cells, Ku loading is limited to ∼1-2 molecules per DNA end. The mechanisms enforcing this limit are currently unclear. Here, we show that the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), but not its protein kinase activity, is required to prevent excessive Ku entry into chromatin. Ku accumulation is further restricted by two mechanisms: a neddylation/FBXL12-dependent process that actively removes loaded Ku molecules throughout the cell cycle and a CtIP/ATM-dependent mechanism that operates in S phase. Finally, we demonstrate that the misregulation of Ku loading leads to impaired transcription in the vicinity of DNA ends. Together, our data shed light on the multiple mechanisms operating to prevent Ku from invading chromatin and interfering with other DNA transactions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Crosses between closely related species of frogs result in inviable embryos that die following catastrophic cell divisions. A new study identifies defects in centromere maintenance and DNA replication stress as key forces driving this incompatibility. [ABSTRACT FROM AUTHOR]

Cell cycle control relies on a delicate balance of phosphorylation with CDK1 and phosphatases like PP1 and PP2A-B55. Yet, identifying the primary substrate responsible for cell cycle oscillations remains a challenge. We uncover the pivotal role of phospho-regulation in the anaphase-promoting complex/cyclosome (APC/C), particularly through the Apc1-loop 300 domain (Apc1-300L), orchestrated by CDK1 and PP2A-B55. Premature activation of PP2A-B55 during mitosis, induced by Greatwall kinase depletion, leads to Apc1-300L dephosphorylation, stalling APC/C activity and delaying Cyclin B degradation. This effect can be counteracted using the B55-specific inhibitor pEnsa or by removing Apc1-300L. We also show Cdc20's dynamic APC/C interaction across cell cycle stages, but dephosphorylation of Apc1-300L specifically inhibits further Cdc20 recruitment. Our study underscores APC/C's central role in cell cycle oscillation, identifying it as a primary substrate regulated by the CDK-PP2A partnership., Competing Interests: Declaration of interests The authors declare no competing interests., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

Functional studies in post-embryonic Xenopus tadpoles are challenging because embryonic perturbations often lead to developmental consequences, such as lethality. Here, we describe a high-throughput protocol for tail vein injection to introduce fluorescent tracers into tadpoles, which we have previously used to effectively inject morpholinos and molecular antagonists. We describe steps for safely positioning tadpoles onto agarose double-coated plates, draining media, injecting into the ventral tail vein, rehydrating plates, and sorting tadpoles by fluorescence with minimal injury for high-throughput experiments. For complete details on the use and execution of this protocol, please refer to Kakebeen et al., 1 Patel et al., 2 and Patel et al. 3 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The development of hindlimbs in tetrapod species relies specifically on the transcription factor TBX4. In humans, heterozygous loss-of-function TBX4 mutations cause dominant small patella syndrome (SPS) due to haploinsufficiency. Here, we characterize a striking clinical entity in four fetuses with complete posterior amelia with pelvis and pulmonary hypoplasia (PAPPA). Through exome sequencing, we find that PAPPA syndrome is caused by homozygous TBX4 inactivating mutations during embryogenesis in humans. In two consanguineous couples, we uncover distinct germline TBX4 coding mutations, p.Tyr113∗ and p.Tyr127Asn, that segregated with SPS in heterozygous parents and with posterior amelia with pelvis and pulmonary hypoplasia syndrome (PAPPAS) in one available homozygous fetus. A complete absence of TBX4 transcripts in this proband with biallelic p.Tyr113∗ stop-gain mutations revealed nonsense-mediated decay of the endogenous mRNA. CRISPR/Cas9-mediated TBX4 deletion in Xenopus embryos confirmed its restricted role during leg development. We conclude that SPS and PAPPAS are allelic diseases of TBX4 deficiency and that TBX4 is an essential transcription factor for organogenesis of the lungs, pelvis, and hindlimbs in humans. [ABSTRACT FROM AUTHOR]

Convergent extension (CE) requires the coordinated action of the planar cell polarity (PCP) proteins 1,2 and the actin cytoskeleton, 3,4,5,6 but this relationship remains incompletely understood. For example, PCP signaling orients actomyosin contractions, yet actomyosin is also required for the polarized localization of PCP proteins. 7,8 Moreover, the actin-regulating Septins play key roles in actin organization 9 and are implicated in PCP and CE in frogs, mice, and fish 5,6,10,11,12 but execute only a subset of PCP-dependent cell behaviors. Septin loss recapitulates the severe tissue-level CE defects seen after core PCP disruption yet leaves overt cell polarity intact. 5 Together, these results highlight the general fact that cell movement requires coordinated action by distinct but integrated actin populations, such as lamella and lamellipodia in migrating cells 13 or medial and junctional actin populations in cells engaged in apical constriction. 14,15 In the context of Xenopus mesoderm CE, three such actin populations are important, a superficial meshwork known as the "node-and-cable" system, 4,16,17,18 a contractile network at deep cell-cell junctions, 6,19 and mediolaterally oriented actin-rich protrusions, which are present both superficially and deeply. 4,19,20,21 Here, we exploited the amenability of the uniquely "two-dimensional" node and cable system to probe the relationship between PCP proteins, Septins, and the polarization of this actin network. We find that the PCP proteins Vangl2 and Prickle2 and Septins co-localize at nodes, and that the node and cable system displays a cryptic, PCP- and Septin-dependent anteroposterior (AP) polarity in its organization and dynamics. • PCP proteins localize to the actin node and cable system during Xenopus gastrulation • The node and cable system displays a cryptic anteroposterior polarity • Anteroposterior polarity of the node and cable system is PCP and Septin dependent • Sept7 controls local dynamics of polarized actin flow Devitt et al. explore the interplay of PCP proteins, actin, and Septins during convergent extension, revealing new complexity to the polarization of the actin cytoskeleton during gastrulation. [ABSTRACT FROM AUTHOR]

Oocytes are arrested in prophase I. In vertebrates, meiotic resumption is triggered by hormonal stimulation that results in cAMP-dependent protein kinase (PKA) downregulation leading to Cdk1 activation. Yet the pathways connecting PKA to Cdk1 remain unclear. Here, we identify molecular events triggered by PKA downregulation occurring upstream of Cdk1 activation. We describe a two-step regulation controlling cyclin B1 and Mos accumulation, which depends on both translation and stabilization. Cyclin B1 accumulation is triggered by PKA inhibition upstream of Cdk1 activation, while its translation requires Cdk1 activity. Conversely, Mos translation initiates in response to the hormone, but the protein accumulates only downstream of Cdk1. Furthermore, two successive translation waves take place, the first controlled by PKA inhibition and the second by Cdk1 activation. Notably, Arpp19, an essential PKA effector, does not regulate the early PKA-dependent events. This study elucidates how PKA downregulation orchestrates multiple pathways that converge toward Cdk1 activation and induce the oocyte G2/M transition., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Morphogenetic movements during animal development involve repeated making and breaking of cell-cell contacts. Recent biophysical models of cell-cell adhesion integrate adhesion molecule interactions and cortical cytoskeletal tension modulation, describing equilibrium states for established contacts. We extend this emerging unified concept of adhesion to contact formation kinetics, showing that aggregating Xenopus embryonic cells rapidly achieve Ca 2+ -independent low-contact states. Subsequent transitions to cadherin-dependent high-contact states show rapid decreases in contact cortical F-actin levels but slow contact area growth. We developed a biophysical model that predicted contact growth quantitatively from known cellular and cytoskeletal parameters, revealing that elastic resistance to deformation and cytoskeletal network turnover are essential determinants of adhesion kinetics. Characteristic time scales of contact growth to low and high states differ by an order of magnitude, being at a few minutes and tens of minutes, respectively, thus providing insight into the timescales of cell-rearrangement-dependent tissue movements., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The oocyte-to-embryo transition (OET) is regulated by maternal products stored in the oocyte cytoplasm, independent of transcription. How maternal products are precisely remodeled to dictate the OET remains largely unclear. In this work, we discover the dynamic solubility phase transition of maternal RNAs during Xenopus OET. We have identified 863 maternal transcripts that transition from a soluble state to a detergent-insoluble one after oocyte maturation. These RNAs are enriched in the animal hemisphere, and many of them encode key cell cycle regulators. In contrast, 165 transcripts, including nearly all Xenopus germline RNAs and some vegetally localized somatic RNAs, undergo an insoluble-to-soluble phase transition. This phenomenon is conserved in zebrafish. Our results demonstrate that the phase transition of germline RNAs influences their susceptibility to RNA degradation machinery and is mediated by the remodeling of germ plasm. This work thus identifies important remodeling mechanisms that act on RNAs to control vertebrate OET., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

SOX4, together with SOX11 and SOX12, forms group C of SRY-related (SOX) transcription factors. They play key roles, often in redundancy, in multiple developmental pathways, including neurogenesis and skeletogenesis. De novo SOX11 heterozygous mutations have been shown to cause intellectual disability, growth deficiency, and dysmorphic features compatible with mild Coffin-Siris syndrome. Using trio-based exome sequencing, we here identify de novo SOX4 heterozygous missense variants in four children who share developmental delay, intellectual disability, and mild facial and digital morphological abnormalities. SOX4 is highly expressed in areas of active neurogenesis in human fetuses, and sox4 knockdown in Xenopus embryos diminishes brain and whole-body size. The SOX4 variants cluster in the highly conserved, SOX family-specific HMG domain, but each alters a different residue. In silico tools predict that each variant affects a distinct structural feature of this DNA-binding domain, and functional assays demonstrate that these SOX4 proteins carrying these variants are unable to bind DNA in vitro and transactivate SOX reporter genes in cultured cells. These variants are not found in the gnomAD database of individuals with presumably normal development, but 12 other SOX4 HMG-domain missense variants are recorded and all demonstrate partial to full activity in the reporter assay. Taken together, these findings point to specific SOX4 HMG-domain missense variants as the cause of a characteristic human neurodevelopmental disorder associated with mild facial and digital dysmorphism. [ABSTRACT FROM AUTHOR]

An instructive role for metabolism in embryonic patterning is emerging, although a role for mitochondria is poorly defined. We demonstrate that mitochondrial oxidative metabolism establishes the embryonic patterning center, the Spemann-Mangold Organizer, via hypoxia-inducible factor 1α (Hif-1α) in Xenopus. Hypoxia or decoupling ATP production from oxygen consumption expands the Organizer by activating Hif-1α. In addition, oxygen consumption is 20% higher in the Organizer than in the ventral mesoderm, indicating an elevation in mitochondrial respiration. To reconcile increased mitochondrial respiration with activation of Hif-1α, we discovered that the "free" c-subunit ring of the F 1 F o  ATP synthase creates an inner mitochondrial membrane leak, which decouples ATP production from respiration at the Organizer, driving Hif-1α activation there. Overexpression of either the c-subunit or Hif-1α is sufficient to induce Organizer cell fates even when β-catenin is inhibited. We propose that mitochondrial leak metabolism could be a general mechanism for activating Hif-1α and Wnt signaling., Competing Interests: Declaration of interests M.K.K. is a founder of Victory Genomics Inc., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Summary Maternally inherited noncoding RNAs (ncRNAs) can regulate zygotic gene expression across generations [ 1–4 ]. Recently, many stable intronic sequence RNAs (sisRNAs), which are byproducts of pre-mRNA splicing, were found to be maternally deposited and persist till zygotic transcription in Xenopus and Drosophila [ 5–7 ]. In various organisms, sisRNAs can be in linear or circular conformations, and they have been suggested to regulate host gene expression [ 5–10 ]. It is unknown whether maternally deposited sisRNAs can regulate zygotic gene expression in the embryos. Here, we show that a maternally inherited sisRNA ( sisR-4 ) from the deadpan locus is important for embryonic development in Drosophila . Mothers, but not fathers, mutant for sisR-4 produce embryos that fail to hatch. During embryogenesis, sisR-4 promotes transcription of its host gene ( deadpan ), which is essential for development. Interestingly, sisR-4 functions by activating an enhancer present in the intron where sisR-4 is encoded. We propose that a maternal sisRNA triggers expression of its host gene via a positive feedback loop during embryogenesis. [ABSTRACT FROM AUTHOR]

Summary Conjoined twins fused at the thorax display an enigmatic left-right defect: although left twins are normal, laterality is disturbed in one-half of right twins [ 1–3 ]. Molecularly, this randomization corresponds to a lack of asymmetric Nodal cascade induction in right twins [ 4 ]. We studied leftward flow [ 5, 6 ] at the left-right organizer (LRO) [ 7, 8 ] in thoracopagus twins in Xenopus , which displayed a duplicated, fused, and ciliated LRO. Cilia were motile and produced a leftward flow from the right LRO margin of the right to the left margin of the left twin. Motility was required for correct laterality in left twins, as knockdown of dynein motor dnah9 prevented Nodal cascade induction. Nodal was rescued by parallel knockdown of the inhibitor dand5 [ 9, 10 ] on the left side of the left twin. Lack of Nodal induction in the right twin, despite the presence of flow, was due to insufficient suppression of dand5 . Knockdown of dand5 at the center of the fused LRO resulted in asymmetric Nodal cascade induction in the right twin as well. Manipulation of leftward flow and dand5 in a targeted and sided manner induced the Nodal cascade in a predictable manner, in the left twin, the right one, both, or neither. Laterality in conjoined twins thus was determined by cilia-driven leftward fluid flow like in single embryos, which solves a century-old riddle, as the phenomenon was already studied by some of the founders of experimental embryology, including Dareste [ 11 ], Fol and Warynsky [ 12 ], and Spemann and Falkenberg [ 13 ] (reviewed in [ 14 ]). [ABSTRACT FROM AUTHOR]

Although polyploidization is frequent in development, cancer, and evolution, impacts on animal metabolism are poorly understood. In Xenopus frogs, the number of genome copies (ploidy) varies across species and can be manipulated within a species. Here, we show that triploid tadpoles contain fewer, larger cells than diploids and consume oxygen at a lower rate. Drug treatments revealed that the major processes accounting for tadpole energy expenditure include cell proliferation, biosynthesis, and maintenance of plasma membrane potential. While inhibiting cell proliferation did not abolish the oxygen consumption difference between diploids and triploids, treatments that altered cellular biosynthesis or electrical potential did. Combining these results with a simple mathematical framework, we propose that the decrease in total cell surface area lowered production and activity of plasma membrane components including the Na+/K+ ATPase, reducing energy consumption in triploids. Comparison of Xenopus species that evolved through polyploidization revealed that metabolic differences emerged during development when cell size scaled with genome size. Thus, ploidy affects metabolism by altering the cell surface area to volume ratio in a multicellular organism. [Display omitted] • Xenopus embryos show a power-law scaling relationship of metabolic rate with mass • Triploid embryos have fewer, larger cells than diploids, reducing metabolic rate • Energetic costs of the plasma membrane maintenance are lowered in triploid cells • Metabolic rate scales with cell size, not genome size across three Xenopus species Cadart et al. show that triploid X. laevis tadpoles have fewer, larger cells and a lower oxygen consumption rate than diploids due to reduced total cell surface area, which decreases energy required to maintain the plasma membrane. Comparing Xenopus species ranging 6-fold in ploidy reveals that metabolic rate scales with cell size, not genome size. [ABSTRACT FROM AUTHOR]

Genome and cell size are strongly correlated across species 1,2,3,4,5,6 and influence physiological traits like developmental rate. 7,8,9,10,11,12 Although size scaling features such as the nuclear-cytoplasmic (N/C) ratio are precisely maintained in adult tissues, 13 it is unclear when during embryonic development size scaling relationships are established. Frogs of the genus Xenopus provide a model to investigate this question, since 29 extant Xenopus species vary in ploidy from 2 to 12 copies (N) of the ancestral frog genome, ranging from 20 to 108 chromosomes. 14,15 The most widely studied species, X. laevis (4N = 36) and X. tropicalis (2N = 20), scale at all levels, from body size to cellular and subcellular levels. 16 Paradoxically, the rare, critically endangered dodecaploid (12N = 108) Xenopus longipes (X. longipes) is a small frog. 15,17 We observed that despite some morphological differences, X. longipes and X. laevis embryogenesis occurred with similar timing, with genome to cell size scaling emerging at the swimming tadpole stage. Across the three species, cell size was determined primarily by egg size, whereas nuclear size correlated with genome size during embryogenesis, resulting in different N/C ratios in blastulae prior to gastrulation. At the subcellular level, nuclear size correlated more strongly with genome size, whereas mitotic spindle size scaled with cell size. Our cross-species study indicates that scaling of cell size to ploidy is not due to abrupt changes in cell division timing, that different size scaling regimes occur during embryogenesis, and that the developmental program of Xenopus is remarkably consistent across a wide range of genome and egg sizes. • Dodecaploid X. longipes provides a model to examine effects of large genome size • Early developmental timing is similar across species, despite different N/C ratios • Genome to cell size scaling emerges at the swimming tadpole stage • Subcellular structures possess distinct scaling regimes during development Miller et al. examine the development of frog species of varying genome and egg sizes and observe that embryogenesis is characterized by distinct scaling regimes at the cellular and subcellular levels. Developmental timing is similar, despite differing nuclear-cytoplasmic ratios, with genome to cell size scaling emerging at the tadpole stage. [ABSTRACT FROM AUTHOR]

Summary Blastoporal expression of the T-box transcription factor gene brachyury is conserved in most metazoans [ 1, 2 ]. Its role in mesoderm formation has been intensively studied in vertebrates [ 3–6 ]. However, its fundamental function near the blastopore is poorly understood in other phyla. Cnidarians are basal metazoans that are important for understanding evolution of metazoan body plans [ 7, 8 ]. Because they lack mesoderm, they have been used to investigate the evolutionary origins of mesoderm [ 1, 9–11 ]. Here, we focus on corals, a primitive clade of cnidarians that diverged from sea anemones ∼500 mya [ 12 ]. We developed a microinjection method for coral eggs to examine Brachyury functions during embryogenesis of the scleractinian coral, Acropora digitifera . Because Acropora embryos undergo pharynx formation after the blastopore closes completely [ 13–15 ], they are useful to understand Brachyury functions in gastrulation movement and pharynx formation. We show that blastoporal expression of brachyury is directly activated by Wnt/β-catenin signaling in the ectoderm of coral embryos, indicating that the regulatory axis from Wnt/β-catenin signaling to brachyury is highly conserved among eumetazoans. Loss-of-function analysis demonstrated that Brachyury is required for pharynx formation but not for gastrulation movement. Genome-wide transcriptome analysis demonstrated that genes positively regulated by Brachyury are expressed in the ectoderm of Acropora gastrulae, while negatively regulated genes are in endoderm. Therefore, germ layer demarcation around the blastopore appears to be the evolutionarily conserved role of Brachyury during gastrulation. Compared with Brachyury functions in vertebrate mesoderm-ectoderm and mesoderm-endoderm demarcation [ 4–6 ], our results suggest that the vertebrate-type mesoderm may have originated from brachyury -expressing ectoderm adjacent to endoderm. [ABSTRACT FROM AUTHOR]

Summary Higher plants take up nutrients via the roots and load them into xylem vessels for translocation to the shoot. After uptake, anions have to be channeled toward the root xylem vessels. Thereby, xylem parenchyma and pericycle cells control the anion composition of the root-shoot xylem sap [ 1–6 ]. The fact that salt-tolerant genotypes possess lower xylem-sap Cl − contents compared to salt-sensitive genotypes [ 7–10 ] indicates that membrane transport proteins at the sites of xylem loading contribute to plant salinity tolerance via selective chloride exclusion. However, the molecular mechanism of xylem loading that lies behind the balance between NO 3 − and Cl − loading remains largely unknown. Here we identify two root anion channels in Arabidopsis , SLAH1 and SLAH3, that control the shoot NO 3 − /Cl − ratio. The AtSLAH1 gene is expressed in the root xylem-pole pericycle, where it co-localizes with AtSLAH3 . Under high soil salinity, AtSLAH1 expression markedly declined and the chloride content of the xylem sap in AtSLAH1 loss-of-function mutants was half of the wild-type level only. SLAH3 anion channels are not active per se but require extracellular nitrate and phosphorylation by calcium-dependent kinases (CPKs) [ 11–13 ]. When co-expressed in Xenopus oocytes, however, the electrically silent SLAH1 subunit gates SLAH3 open even in the absence of nitrate- and calcium-dependent kinases. Apparently, SLAH1/SLAH3 heteromerization facilitates SLAH3-mediated chloride efflux from pericycle cells into the root xylem vessels. Our results indicate that under salt stress, plants adjust the distribution of NO 3 − and Cl − between root and shoot via differential expression and assembly of SLAH1/SLAH3 anion channel subunits. [ABSTRACT FROM AUTHOR]

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]

Although central to evolution, the causes of hybrid inviability that drive reproductive isolation are poorly understood. Embryonic lethality occurs when the eggs of the frog X. tropicalis are fertilized with either X. laevis or X. borealis sperm. We observed that distinct subsets of paternal chromosomes failed to assemble functional centromeres, causing their mis-segregation during embryonic cell divisions. Core centromere DNA sequence analysis revealed little conservation among the three species, indicating that epigenetic mechanisms that normally operate to maintain centromere integrity are disrupted on specific paternal chromosomes in hybrids. In vitro reactions combining X. tropicalis egg extract with either X. laevis or X. borealis sperm chromosomes revealed that paternally matched or overexpressed centromeric histone CENP-A and its chaperone HJURP could rescue centromere assembly on affected chromosomes in interphase nuclei. However, although the X. laevis chromosomes maintained centromeric CENP-A in metaphase, X. borealis chromosomes did not and also displayed ultra-thin regions containing ribosomal DNA. Both centromere assembly and morphology of X. borealis mitotic chromosomes could be rescued by inhibiting RNA polymerase I or preventing the collapse of stalled DNA replication forks. These results indicate that specific paternal centromeres are inactivated in hybrids due to the disruption of associated chromatin regions that interfere with CENP-A incorporation, at least in some cases due to conflicts between replication and transcription machineries. Thus, our findings highlight the dynamic nature of centromere maintenance and its susceptibility to disruption in vertebrate interspecies hybrids. [Display omitted] • Divergent core centromeric sequences do not underlie Xenopus hybrid inviability • CENP-A eviction from specific paternal chromosomes requires cell cycle progression • Driving assembly can rescue and maintain X. laevis , but not X. borealis , centromeres • Mitotic replication stress and conflicts lead to X. borealis chromosome defects Centromere incompatibilities in inviable Xenopus hybrids are DNA sequence independent and result from the disruption of epigenetic pathways required for CENP-A maintenance. [ABSTRACT FROM AUTHOR]

Topological stress can cause converging replication forks to stall during termination of vertebrate DNA synthesis. However, replication forks ultimately overcome fork stalling, suggesting that alternative mechanisms of termination exist. Using proteomics in Xenopus egg extracts, we show that the helicase RTEL1 and the replisome protein MCM10 are highly enriched on chromatin during fork convergence and are crucially important for fork convergence under conditions of topological stress. RTEL1 and MCM10 cooperate to promote fork convergence and do not impact topoisomerase activity but do promote fork progression through a replication barrier. Thus, RTEL1 and MCM10 play a general role in promoting progression of stalled forks, including when forks stall during termination. Our data reveal an alternate mechanism of termination involving RTEL1 and MCM10 that can be used to complete DNA synthesis under conditions of topological stress., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Asymmetric localization of mRNAs is crucial for cell polarity and cell fate determination. By performing fractionation RNA-seq, we report here that a large number of maternal RNAs are associated with the ER in Xenopus oocytes but are released into the cytosol after oocyte maturation. We provide evidence that the majority of ER-associated RNA-binding proteins (RBPs) remain associated with the ER after oocyte maturation. However, all ER-associated RBPs analyzed exhibit reduced binding to some of their target RNAs after oocyte maturation. Our results further show that the ER is remodeled massively during oocyte maturation, leading to the formation of a widespread tubular ER network in the animal hemisphere that is required for the asymmetric localization of mRNAs in mature eggs. Thus, our findings demonstrate that dynamic regulation of RNA-ER association and remodeling of the ER are important for the asymmetric localization of RNAs during development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Despite the fundamental roles of TGF-β family signaling in cell fate determination in all metazoans, the mechanism by which these signals are spatially and temporally interpreted remains elusive. The cell-context-dependent function of TGF-β signaling largely relies on transcriptional regulation by SMAD proteins. Here, we discover that the DNA repair-related protein, HMCES, contributes to early development by maintaining nodal/activin- or BMP-signaling-regulated transcriptional network. HMCES binds with R-SMAD proteins, co-localizing at active histone marks. However, HMCES chromatin occupancy is independent on nodal/activin or BMP signaling. Mechanistically, HMCES competitively binds chromatin to limit binding by R-SMAD proteins, thereby forcing their dissociation and resulting in repression of their regulatory effects. In Xenopus laevis embryo, hmces KD causes dramatic development defects with abnormal left-right axis asymmetry along with increasing expression of lefty1. These findings reveal HMCES transcriptional regulatory function in the context of TGF-β family signaling., Competing Interests: Declaration of interests The authors declare no competing interest., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

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: In vertebrates, most inner organs are asymmetrically arranged with respect to the main body axis []. Symmetry breakage in fish, amphibian, and mammalian embryos depends on cilia-driven leftward flow of extracellular fluid during neurulation []. Flow induces the asymmetric nodal cascade that governs asymmetric organ morphogenesis and placement []. In the frog Xenopus, an alternative laterality-generating mechanism involving asymmetric localization of serotonin at the 32-cell stage has been proposed []. However, no functional linkage between this early localization and flow at neurula stage has emerged. Here, we report that serotonin signaling is required for specification of the superficial mesoderm (SM), which gives rise to the ciliated gastrocoel roof plate (GRP) where flow occurs []. Flow and asymmetry were lost in embryos in which serotonin signaling was downregulated. Serotonin, which we found uniformly distributed along the main body axes in the early embryo, was required for Wnt signaling, which provides the instructive signal to specify the GRP. Importantly, serotonin was required for Wnt-induced double-axis formation as well. Our data confirm flow as primary mechanism of symmetry breakage and suggest a general role of serotonin as competence factor for Wnt signaling during axis formation in Xenopus. [ABSTRACT FROM AUTHOR]

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: Bistability of the Cdk1-Wee1-Cdc25 mitotic control network underlies the switch-like transitions between interphase and mitosis. Here, we show by mathematical modeling and experiments in Xenopus egg extracts that protein phosphatase 2A (PP2A), which can dephosphorylate Cdk1 substrates, is essential for this bistability. PP2A inhibition in early interphase abolishes the switch-like response of the system to Cdk1 activity, promoting mitotic onset even with very low levels of Cyclin, Cdk1, and Cdc25, while simultaneously inhibiting DNA replication. Furthermore, even if replication has already initiated, it cannot continue in mitosis. Exclusivity of S and M phases does not depend on bistability only, since partial PP2A inhibition prevents replication without inducing mitotic onset. In these conditions, interphase-level mitotic kinases inhibit Cyclin E-Cdk2 chromatin loading, blocking initiation complex formation. Therefore, by counteracting both Cdk1 activation and activity of mitotic kinases, PP2A ensures robust separation of S phase and mitosis and dynamic transitions between the two states. [ABSTRACT FROM AUTHOR]

Summary: Faithful transmission of genomic information requires tight spatiotemporal regulation of DNA replication factors. In the licensing step of DNA replication, CDT-1 is loaded onto chromatin to subsequently promote the recruitment of additional replication factors, including CDC-45 and GINS. During the elongation step, the CDC-45/GINS complex moves with the replication fork; however, it is largely unknown how its chromatin association is regulated. Here, we show that the chaperone-like ATPase CDC-48/p97 coordinates degradation of CDT-1 with release of the CDC-45/GINS complex. C. elegans embryos lacking CDC-48 or its cofactors UFD-1/NPL-4 accumulate CDT-1 on mitotic chromatin, indicating a critical role of CDC-48 in CDT-1 turnover. Strikingly, CDC-48UFD-1/NPL-4-deficient embryos show persistent chromatin association of CDC-45/GINS, which is a consequence of CDT-1 stabilization. Moreover, our data confirmed a similar regulation in Xenopus egg extracts, emphasizing a conserved coordination of licensing and elongation events during eukaryotic DNA replication by CDC-48/p97. [ABSTRACT FROM AUTHOR]

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: In the developing Xenopus tadpole, conditioning with 20 min of visual stimulation leads to increased proBDNF protein levels in the tectum measured 4 hr later. Following conditioning, the ability to induce direction selectivity in tectal neurons, as well as both retinotectal long-term potentiation and depression, thought to underlie this phenomenon, was strongly facilitated. This facilitation was blocked by knockdown of BDNF expression in tectal neurons. Animals that had been exposed to visual conditioning and subsequently received normal visual input for 7–11 hr exhibited higher spatial frequency thresholds of tectal cell responses to counterphasing gratings than nonconditioned control animals. An improvement in visual acuity was confirmed by enhanced sensitivity to counterphasing gratings in a behavioral test. These results indicate that brief sensory stimulation, by initiating nuclear transcription and de novo protein synthesis of BDNF, can facilitate the refinement of response properties in the developing visual system. [ABSTRACT FROM AUTHOR]

Summary: Disruptions of anatomical left-right asymmetry result in life-threatening heterotaxic birth defects in vital organs. We performed a small molecule screen for left-right asymmetry phenotypes in Xenopus embryos and discovered a pyridine analog, heterotaxin, which disrupts both cardiovascular and digestive organ laterality and inhibits TGF-β-dependent left-right asymmetric gene expression. Heterotaxin analogs also perturb vascular development, melanogenesis, cell migration, and adhesion, and indirectly inhibit the phosphorylation of an intracellular mediator of TGF-β signaling. This combined phenotypic profile identifies these compounds as a class of TGF-β signaling inhibitors. Notably, heterotaxin analogs also possess highly desirable antitumor properties, inhibiting epithelial-mesenchymal transition, angiogenesis, and tumor cell proliferation in mammalian systems. Our results suggest that assessing multiple organ, tissue, cellular, and molecular parameters in a whole organism context is a valuable strategy for identifying the mechanism of action of bioactive compounds. [Copyright &y& Elsevier]

Summary: Cdc25C is a critical component of the interlinked positive and double-negative feedback loops that constitute the bistable mitotic trigger. Computational studies have indicated that the trigger''s bistability should be more robust if the individual legs of the loops exhibit ultrasensitive responses. Here, we show that in Xenopus extracts two measures of Cdc25C activation (hyperphosphorylation and Ser 287 dephosphorylation) are highly ultrasensitive functions of the Cdk1 activity; estimated Hill coefficients were 11 to 32. Some of Cdc25C''s ultrasensitivity can be reconstituted in vitro with purified components, and the reconstituted ultrasensitivity depends upon multisite phosphorylation. The response functions determined here for Cdc25C and previously for Wee1A allow us to formulate a simple mathematical model of the transition between interphase and mitosis. The model shows how the continuously variable regulators of mitosis work collectively to generate a switch-like, hysteretic response. [Copyright &y& Elsevier]

Summary: The duplication of eukaryotic genomes involves the replication of DNA from multiple origins of replication. In S phase, two sister replisomes assemble at each active origin, and they replicate DNA in opposite directions. Little is known about the functional relationship between sister replisomes. Some data imply that they travel away from one another and thus function independently. Alternatively, sister replisomes may form a stationary, functional unit that draws parental DNA toward itself. If this “double replisome” model is correct, a constrained DNA molecule should not undergo replication. To test this prediction, lambda DNA was stretched and immobilized at both ends within a microfluidic flow cell. Upon exposure to Xenopus egg extracts, this DNA underwent extensive replication by a single pair of diverging replisomes. The data show that there is no obligatory coupling between sister replisomes and, together with other studies, imply that genome duplication involves autonomously functioning replisomes. [ABSTRACT FROM AUTHOR]

Summary: Regulation of progenitor cell fate determines the numbers of neurons in the developing brain. While proliferation of neural progenitors predominates during early central nervous system (CNS) development, progenitor cell fate shifts toward differentiation as CNS circuits develop, suggesting that signals from developing circuits may regulate proliferation and differentiation. We tested whether activity regulates neurogenesis in vivo in the developing visual system of Xenopus tadpoles. Both cell proliferation and the number of musashi1-immunoreactive progenitors in the optic tectum decrease as visual system connections become stronger. Visual deprivation for 2 days increased proliferation of musashi1-immunoreactive radial glial progenitors, while visual experience increased neuronal differentiation. Morpholino-mediated knockdown and overexpression of musashi1 indicate that musashi1 is necessary and sufficient for neural progenitor proliferation in the CNS. These data demonstrate a mechanism by which increased brain activity in developing circuits decreases cell proliferation and increases neuronal differentiation through the downregulation of musashi1 in response to circuit activity. [ABSTRACT FROM AUTHOR]

Summary: H1 “linker” histones bind dynamically to nucleosomes and promote their compaction into chromatin fibers []. Developmental H1 isoforms are evolutionarily conserved, but their function, regulation, and posttranslational modifications are poorly understood []. In Xenopus egg extracts, the embryonic linker histone H1M does not affect nuclear assembly or replication but is required for proper chromosome architecture during mitosis []. We report here that somatic H1 isoforms, which are more positively charged and feature multiple Cdk1 phosphorylation sites, cannot substitute for H1M at endogenous concentrations, instead causing chromatin compaction during interphase and dissociating from chromosomes at the onset of mitosis. Mitotic Cdk1 phosphorylation is not responsible for this dissociation and instead functions to enhance H1 binding in egg extracts and embryos. Nuclear import receptors RanBP7 and importin β bind tightly to somatic H1 but not H1M, and addition of a constitutively active Ran mutant abolishes this interaction and enhances the ability of somatic H1 to rescue mitotic chromosome architecture. Our results reveal distinct regulatory mechanisms among linker histone isoforms and a specific role for H1M to compact chromosomes during egg meiotic arrest and early embryonic divisions. [Copyright &y& Elsevier]

Summary: Vertebrate laterality, which is manifested by asymmetrically placed organs [], depends on asymmetric activation of the Nodal signaling cascade in the left lateral plate mesoderm []. In fish, amphibians, and mammals, a cilia-driven leftward flow of extracellular fluid acts upstream of the Nodal cascade []. The direct target of flow has remained elusive. In Xenopus, flow occurs at the gastrocoel roof plate (GRP) in the dorsal midline of the embryo []. The GRP is bordered by a second, bilaterally symmetrical Nodal expression domain []. Here we identify the Nodal inhibitor Coco as a critical target of flow. Coco and Xenopus Nodal-related 1 (Xnr1) are coexpressed in the lateralmost ciliated GRP cells. Coco becomes downregulated on the left side of the GRP as a direct readout of flow. Ablation of flow prevented Coco repression, whereas Xnr1 expression was independent of flow. Loss of flow-induced laterality defects were rescued by knockdown of Coco on the left side. Parallel knockdown of Coco and Xnr1 in GRP cells restored laterality defects in flow-impaired embryos, demonstrating that Coco acted through GRP-expressed Xnr1. Coco thus acts as a critical target of flow, suggesting that symmetry is broken by flow-mediated left-asymmetric release of Nodal repression at the midline. [Copyright &y& Elsevier]

Summary: Ephrin-B/EphB and Wnts are known to regulate synapse maturation and plasticity, besides serving as axon guidance molecules, but the relevance of such synaptic regulation to neural circuit functions in vivo remains unclear. In this study, we have examined the role of ephrin-B and Wnt signaling in regulating visual experience-dependent and developmental plasticity of receptive fields (RFs) of tectal cells in the developing Xenopus optic tectum. We found that repetitive exposure to unidirectional moving visual stimuli caused varying degrees of shift in the RFs in different regions of the tectum. By acute perfusion of exogenous antagonists and inducible transgene expression, we showed that ephrin-B signaling in presynaptic retinal ganglion cells and Wnt secretion from tectal cells are specifically responsible for the enhanced visual stimulation-induced changes in neuronal responses and RFs in the ventral and dorsal tectum, respectively. Thus, ephrin-B and Wnt signaling contribute to region-specific plasticity of visual circuit functions. [Copyright &y& Elsevier]

Summary: Regulated protein degradation via the ubiquitin-proteasome system (UPS) plays a central role in building synaptic connections, yet little is known about either which specific UPS components are involved or UPS targets in neurons. We report that inhibiting the UPS in developing Xenopus retinal ganglion cells (RGCs) with a dominant-negative ubiquitin mutant decreases terminal branching in the tectum but does not affect long-range navigation to the tectum. We identify Nedd4 as a prominently expressed E3 ligase in RGC axon growth cones and show that disrupting its function severely inhibits terminal branching. We further demonstrate that PTEN, a negative regulator of the PI3K pathway, is a key downstream target of Nedd4: not only does Nedd4 regulate PTEN levels in RGC growth cones, but also, the decrease of PTEN rescues the branching defect caused by Nedd4 inhibition. Together our data suggest that Nedd4-regulated PTEN is a key regulator of terminal arborization in vivo. [Copyright &y& Elsevier]

Summary: In response to DNA double-strand breaks (DSBs), cells sense the DNA lesions and then activate the protein kinase ATM. Subsequent DSB resection produces RPA-coated ssDNA that is essential for activation of the DNA damage checkpoint and DNA repair by homologous recombination (HR). However, the biochemical mechanism underlying the transition from DSB sensing to resection remains unclear. Using Xenopus egg extracts and human cells, we show that the tumor suppressor protein CtIP plays a critical role in this transition. We find that CtIP translocates to DSBs, a process dependent on the DSB sensor complex Mre11-Rad50-NBS1, the kinase activity of ATM, and a direct DNA-binding motif in CtIP, and then promotes DSB resection. Thus, CtIP facilitates the transition from DSB sensing to processing: it does so by binding to the DNA at DSBs after DSB sensing and ATM activation and then promoting DNA resection, leading to checkpoint activation and HR. [Copyright &y& Elsevier]

Summary: During embryogenesis, brain neurons receiving the same sensory input may undergo potentiation or depression. While the origin of variable plasticity in vivo is unknown, it plays a key role in shaping dynamic neural circuit refinement. Here, we investigate effects of natural visual stimuli on neuronal firing within the intact, awake, developing brain using calcium imaging of 100 s of central neurons in the Xenopus retinotectal system. We find that specific patterns of visual stimuli shift population responses toward either potentiation or depression in an N-methyl-D-aspartate receptor (NMDA-R)-dependent manner. In agreement with Bienenstock-Cooper-Munro metaplasticity, our results show that functional potentiation or depression can be predicted by individual neurons'' specific receptive field properties and historic firing rates. Interestingly, this activity-dependent metaplasticity is itself NMDA-R dependent. Furthermore, network analysis reveals increased correlated firing of neurons that undergo potentiation. These findings implicate metaplasticity as a natural property regulating experience-dependent refinement of nascent embryonic brain circuits. [Copyright &y& Elsevier]

Summary: Cul3, a Cullin family scaffold protein, is thought to mediate the assembly of a large number of SCF (Skp1-Cullin1-F-box protein)-like ubiquitin ligase complexes through BTB domain substrate-recruiting adaptors. Cul3 controls early embryonic development in several genetic models through mechanisms not understood. Very few functional substrate/adaptor pairs for Cul3 ubiquitin ligases have been identified. Here, we show that Cul3 knockdown in human cells results in abnormal actin stress fibers and distorted cell morphology, owing to impaired ubiquitination and degradation of small GTPase RhoA. We identify a family of RhoA-binding BTB domain adaptors conserved from insects to mammals, designated BACURDs. BACURDs form ubiquitin ligase complexes, which selectively ubiquitinate RhoA, with Cul3. Dysfunction of the Cul3/BACURD complex decreases cell migration potential and impairs RhoA-mediated convergent extension movements during Xenopus gastrulation. Our studies reveal a previously unknown mechanism for controlling RhoA degradation and regulating RhoA function in various biological contexts, which involves a Cul3/BACURD ubiquitin ligase complex. [Copyright &y& Elsevier]

Summary: Single cells and multicellular tissues rapidly heal wounds. These processes are considered distinct, but one mode of healing—Rho GTPase-dependent formation and closure of a purse string of actin filaments (F-actin) and myosin-2 around wounds—occurs in single cells and in epithelia . Here, we show that wounding of one cell in Xenopus embryos elicits Rho GTPase activation around the wound and at the nearest cell-cell junctions in the neighbor cells. F-actin and myosin-2 accumulate at the junctions and around the wound itself, and as the resultant actomyosin array closes over the wound site, junctional F-actin and myosin-2 become mechanically integrated with the actin and myosin-2 around the wound, forming a hybrid purse string. When cells are ablated rather than wounded, Rho GTPase activation and F-actin accumulation occur at cell-cell junctions surrounding the ablated cell, and the purse string closes the hole in the epithelium. Elevation of intracellular free calcium, an essential upstream signal for the single-cell wound response , also occurs at the cell-cell contacts and in neighbor cells. Thus, the single and multicellular purse string wound responses represent points on a signaling and mechanical continuum that are integrated by cell-cell junctions. [Copyright &y& Elsevier]

Summary: Substrates of the E3 ubiquitin ligase CRL4Cdt2, including Cdt1 and p21, contain a PCNA-binding motif called a PIP box. Upon binding of the PIP box to PCNA on chromatin, CRL4Cdt2 is recruited and the substrate is ubiquitylated. Importantly, a PIP box cannot be sufficient for destruction, as most PIP box proteins are stable. Using Xenopus egg extracts, we identify two sequence elements in CRL4Cdt2 substrates that promote their proteolysis: a specialized PIP box that confers exceptionally efficient PCNA binding and a basic amino acid 4 residues downstream of the PIP box, which recruits CRL4Cdt2 to the substrate-PCNA complex. We also identify two mechanisms that couple CRL4Cdt2-dependent proteolysis to the chromatin-bound form of PCNA, ensuring that this proteolysis pathway is active only in S phase or after DNA damage. Thus, CRL4Cdt2 recognizes an unusual degron, which is assembled specifically on chromatin via the binding of a specialized PIP box to PCNA. [Copyright &y& Elsevier]