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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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

During oocyte maturation and early embryogenesis, changes in mRNA poly(A)-tail lengths strongly influence translation, but how these tail-length changes are orchestrated has been unclear. Here, we performed tail-length and translational profiling of mRNA reporter libraries (each with millions of 3' UTR sequence variants) in frog oocytes and embryos and in fish embryos. Contrasting to previously proposed cytoplasmic polyadenylation elements (CPEs), we found that a shorter element, UUUUA, together with the polyadenylation signal (PAS), specify cytoplasmic polyadenylation, and we identified contextual features that modulate the activity of both elements. In maturing oocytes, this tail lengthening occurs against a backdrop of global deadenylation and the action of C-rich elements that specify tail-length-independent translational repression. In embryos, cytoplasmic polyadenylation becomes more permissive, and additional elements specify waves of stage-specific deadenylation. Together, these findings largely explain the complex tapestry of tail-length changes observed in early frog and fish development, with strong evidence of conservation in both mice and humans., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Here, we present a protocol for optogenetic dephosphorylation of the phosphoinositide PI(4,5)P 2 at the plasma membrane of Xenopus laevis oocytes. We first describe the co-injection of oocytes with cRNAs encoding (1) a light-activated PI(4,5)P 2 5-phosphatase fusion protein, (2) its dimerization partner fused to the plasma membrane, and (3) the potassium channel reporter for PI(4,5)P 2 dephosphorylation. We then detail blue light illumination to induce PI(4,5)P 2 dephosphorylation, combined with simultaneous two-electrode voltage clamp electrophysiological recording to assess potassium channel current responses. For complete details on the use and execution of this protocol, please refer to Xu et al. (2022). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Peroxisomes are vital metabolic organelles whose matrix enzymes are imported from the cytosol in a folded state by the soluble receptor PEX5. The import mechanism has been challenging to decipher because of the lack of suitable in vitro systems. Here, we present a protocol for reconstituting matrix protein import using Xenopus egg extract. We describe how extract is prepared, how to replace endogenous PEX5 with recombinant versions, and how to perform and interpret a peroxisomal import reaction using a fluorescent cargo. For complete details on the use and execution of this protocol, please refer to Skowyra and Rapoport (2022). 1 ., Competing Interests: Declaration of interests The authors have no competing interests to declare., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Perturbing the temperature of a system modifies its energy landscape, thus providing a ubiquitous tool to understand biological processes. Here, we developed a framework to generate sudden temperature jumps (Tjumps) and sustained temperature steps (Tsteps) to study the temperature dependence of membrane proteins under voltage clamp while measuring the membrane temperature. Utilizing the melanin under the Xenopus laevis oocytes membrane as a photothermal transducer, we achieved short Tjumps up to 9°C in less than 1.5 ms and constant Tsteps for durations up to 150 ms. We followed the temperature at the membrane with sub-ms time resolution by measuring the time course of membrane capacitance, which is linearly related to temperature. We applied Tjumps in Kir1.1 isoform b, which reveals a highly temperature-sensitive blockage relief, and characterized the effects of Tsteps on the temperature-sensitive channels TRPM8 and TRPV1. These newly developed approaches provide a general tool to study membrane protein thermodynamics., Competing Interests: Declaration of interests All other authors declare they have no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The design of an animal's body plan is encoded in the genome, and the execution of this program is a mechanical progression involving coordinated movement of proteins, cells, and whole tissues. Thus, a challenge to understanding morphogenesis is connecting events that occur across various length scales. Here, we describe how a poorly characterized adhesion effector, Arvcf catenin, controls Xenopus head-to-tail axis extension. We find that Arvcf is required for axis extension within the intact organism but not within isolated tissues. We show that the organism-scale phenotype results from a defect in tissue-scale force production. Finally, we determine that the force defect results from the dampening of the pulsatile recruitment of cell adhesion and cytoskeletal proteins to membranes. These results provide a comprehensive understanding of Arvcf function during axis extension and produce an insight into how a cellular-scale defect in adhesion results in an organism-scale failure of development., Competing Interests: Declaration of interests J.B.W. is a member of the Developmental Cell advisory board., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Various types of sensory stimuli have been shown to induce Ca 2+ elevations in glia. However, a mechanistic understanding of the signaling pathways mediating sensory-evoked activity in glia in intact animals is still emerging. During early development of the Xenopus laevis visual system, radial astrocytes in the optic tectum are highly responsive to sensory stimulation. Ca 2+ transients occur spontaneously in radial astrocytes at rest and are abolished by silencing neuronal activity with tetrodotoxin. Visual stimulation drives temporally correlated increases in the activity patterns of neighboring radial astrocytes. Following blockade of all glutamate receptors (gluRs), visually evoked Ca 2+ activity in radial astrocytes persists, while neuronal activity is suppressed. The additional blockade of either glu transporters or sodium-calcium exchangers (NCX) abolishes visually evoked responses in glia. Finally, we demonstrate that blockade of NCX alone is sufficient to prevent visually evoked responses in radial astrocytes, highlighting a pivotal role for NCX in glia during development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Multiciliated cells (MCCs) are extremely highly differentiated, presenting >100 cilia and basal bodies. Therefore, MCC fate is thought to be terminal and irreversible. We analyzed how MCCs are removed from the airway-like mucociliary Xenopus epidermis during developmental tissue remodeling. We found that a subset of MCCs undergoes lateral line-induced apoptosis, but that the majority coordinately trans-differentiate into goblet secretory cells. Both processes are dependent on Notch signaling, while the cellular response to Notch is modulated by Jak/STAT, thyroid hormone, and mTOR signaling. At the cellular level, trans-differentiation is executed through the loss of ciliary gene expression, including foxj1 and pcm1, altered proteostasis, cilia retraction, basal body elimination, as well as the initiation of mucus production and secretion. Our work describes two modes for MCC loss during vertebrate development, the signaling regulation of these processes, and demonstrates that even cells with extreme differentiation features can undergo direct fate conversion., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cell-free extract derived from the eggs of the African clawed frog Xenopus laevis is a well-established model system that has been used historically in bulk aliquots. Here, we describe a microfluidic approach for isolating discrete, biologically relevant volumes of cell-free extract, with more expansive and precise control of extract shape compared with extract-oil emulsions. This approach is useful for investigating the mechanics of intracellular processes affected by cell geometry or cytoplasmic volume, including organelle scaling and positioning mechanisms. For complete details on the use and execution of this protocol, please refer to Geisterfer et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2020 The Authors.)

Zyxin is a cytoskeletal LIM-domain protein that regulates actin cytoskeleton assembly and gene expression. In the present work, we find that zyxin downregulation in Xenopus laevis embryos reduces the expression of numerous genes that regulate cell differentiation, but it enhances that of several genes responsible for embryonic stem cell status, specifically klf4, pou5f3.1, pou5f3.2, pou5f3.3, and vent2.1/2. For pou5f3 family genes (mammalian POU5F1/OCT4 homologs), we show that this effect is the result of mRNA stabilization due to complex formation with the Y-box protein Ybx1. When bound to Ybx1, zyxin interferes with the formation of these complexes, thereby stimulating pou5f3 mRNA degradation. In addition, in zebrafish embryos and human HEK293 cells, zyxin downregulation increases mRNA levels of the pluripotency genes KLF4, NANOG, and POU5F1/OCT4. Our findings indicate that zyxin may play a role as a switch among morphogenetic cell movement, differentiation, and embryonic stem cell status., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Nuclear size plays pivotal roles in gene expression, embryo development, and disease. A central hypothesis in organisms ranging from yeast to vertebrates is that nuclear size scales to cell size. This implies that nuclei may reach steady-state sizes set by limiting cytoplasmic pools of size-regulating components. By monitoring nuclear dynamics in early sea urchin embryos, we found that nuclei undergo substantial growth in each interphase, reaching a maximal size prior to mitosis that declined steadily over the course of development. Manipulations of cytoplasmic volume through multiple chemical and physical means ruled out cell size as a major determinant of nuclear size and growth. Rather, our data suggest that the perinuclear endoplasmic reticulum, accumulated through dynein activity, serves as a limiting membrane pool that sets nuclear surface growth rate. Partitioning of this local pool at each cell division modulates nuclear growth kinetics and dictates size scaling throughout early development., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

To study contributions of N-methyl-D-aspartate receptors (NMDARs) in presynaptic and postsynaptic neurons of the developing visual system, we microinject antisense Morpholino oligonucleotide (MO) against GluN1 into one cell of two-cell-stage Xenopus laevis embryos. The resulting bilateral segregation of MO induces postsynaptic NMDAR (postNMDAR) knockdown in tectal neurons on one side and presynaptic NMDAR (preNMDAR) knockdown in ganglion cells projecting to the other side. PostNMDAR knockdown reduces evoked NMDAR- and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated retinotectal currents. Although the frequency of spontaneous synaptic events is increased, the probability of evoked release is reduced. PreNMDAR knockdown results in larger evoked and unitary synaptic responses. Structurally, postNMDAR and preNMDAR knockdown produce complementary effects. Axonal arbor complexity is reduced by preNMDAR-MO and increased by postNMDAR-MO, whereas tectal dendritic arbors exhibit the inverse. The current study illustrates distinct roles for pre- and postNMDARs in circuit development and reveals extensive transsynaptic regulation of form and function., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The large length scale of Xenopus laevis eggs facilitates observation of bulk cytoplasm dynamics far from the cortex during cytokinesis. The first furrow ingresses through the egg midplane, which is demarcated by chromosomal passenger complex (CPC) localized on microtubule bundles at the boundary between asters. Using an extract system, we found that local kinase activity of the Aurora B kinase (AURKB) subunit of the CPC caused disassembly of F-actin and keratin between asters and local softening of the cytoplasm as assayed by flow patterns. Beads coated with active CPC mimicked aster boundaries and caused AURKB-dependent disassembly of F-actin and keratin that propagated ∼40 μm without microtubules and much farther with microtubules present. Consistent with extract observations, we observed disassembly of the keratin network between asters in zygotes fixed before and during 1 st cytokinesis. We propose that active CPC at aster boundaries locally reduces cytoplasmic stiffness by disassembling actin and keratin networks. Possible functions of this local disassembly include helping sister centrosomes move apart after mitosis, preparing a soft path for furrow ingression, and releasing G-actin from internal networks to build cortical networks that support furrow ingression., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Although immune checkpoint blockers have yielded significant clinical benefits in patients with different malignancies, the efficacy of these therapies is still limited. Here, we show that disruption of transmembrane protein 176B (TMEM176B) contributes to CD8 + T cell-mediated tumor growth inhibition by unleashing inflammasome activation. Lack of Tmem176b enhances the antitumor activity of anti-CTLA-4 antibodies through mechanisms involving caspase-1/IL-1β activation. Accordingly, patients responding to checkpoint blockade therapies display an activated inflammasome signature. Finally, we identify BayK8644 as a potent TMEM176B inhibitor that promotes CD8 + T cell-mediated tumor control and reinforces the antitumor activity of both anti-CTLA-4 and anti-PD-1 antibodies. Thus, pharmacologic de-repression of the inflammasome by targeting TMEM176B may enhance the therapeutic efficacy of immune checkpoint blockers., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The microtubule-based spindle is subjected to various mechanical forces during cell division. How the structure generates and responds to forces while maintaining overall integrity is unknown because we have a poor understanding of the relationship between filament architecture and mechanics. Here, to fill this gap, we combine microneedle-based quantitative micromanipulation with high-resolution imaging, simultaneously analyzing forces and local filament motility in the Xenopus meiotic spindle. We find that microtubules exhibit a compliant, fluid-like mechanical response at the middle of the spindle half, being distinct from those near the pole and the equator. A force altering spindle length induces filament sliding at this compliant array, where parallel microtubules predominate, without influencing equatorial antiparallel filament dynamics. Molecular perturbations suggest that kinesin-5 and dynein contribute to the spindle's local mechanical difference. Together, our data establish a link between spindle architecture and mechanics and uncover the mechanical design of this essential cytoskeletal assembly., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Mechanical forces are essential drivers of numerous biological processes, notably during development. Although it is well recognized that cells sense and adapt to mechanical forces, the signal transduction pathways that underlie mechanosensing have remained elusive. Here, we investigate the impact of mechanical centrifugation force on phosphorylation-mediated signaling in Xenopus embryos. By monitoring temporal phosphoproteome and proteome alterations in response to force, we discover and validate elevated phosphorylation on focal adhesion and tight junction components, leading to several mechanistic insights into mechanosensing and tissue restoration. First, we determine changes in kinase activity profiles during mechanoresponse, identifying the activation of basophilic kinases. Pathway interrogation using kinase inhibitor treatment uncovers a crosstalk between the focal adhesion kinase (FAK) and protein kinase C (PKC) in mechanoresponse. Second, we find LIM domain 7 protein (Lmo7) as upregulated upon centrifugation, contributing to mechanoresponse. Third, we discover that mechanical compression force induces a mesenchymal-to-epithelial transition (MET)-like phenotype., (Copyright © 2019 Elsevier Inc. All rights reserved.)

DNA replication errors generate complex chromosomal rearrangements and thereby contribute to tumorigenesis and other human diseases. One mechanism that triggers these errors is mitotic entry before the completion of DNA replication. To address how mitosis might affect DNA replication, we used Xenopus egg extracts. When mitotic CDK (Cyclin B1-CDK1) is used to drive interphase egg extracts into a mitotic state, the replicative CMG (CDC45/MCM2-7/GINS) helicase undergoes ubiquitylation on its MCM7 subunit, dependent on the E3 ubiquitin ligase TRAIP. Whether replisomes have stalled or undergone termination, CMG ubiquitylation is followed by its extraction from chromatin by the CDC48/p97 ATPase. TRAIP-dependent CMG unloading during mitosis is also seen in C. elegans early embryos. At stalled forks, CMG removal results in fork breakage and end joining events involving deletions and templated insertions. Our results identify a mitotic pathway of global replisome disassembly that can trigger replication fork collapse and DNA rearrangements., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Tight junctions contribute to epithelial barrier function by selectively regulating the quantity and type of molecules that cross the paracellular barrier. Experimental approaches to evaluate the effectiveness of tight junctions are typically global, tissue-scale measures. Here, we introduce Zinc-based Ultrasensitive Microscopic Barrier Assay (ZnUMBA), which we used in Xenopus laevis embryos to visualize short-lived, local breaches in epithelial barrier function. These breaches, or leaks, occur as cell boundaries elongate, correspond to visible breaks in the tight junction, and are followed by transient localized Rho activation, or Rho flares. We discovered that Rho flares restore barrier function by driving concentration of tight junction proteins through actin polymerization and ROCK-mediated localized contraction of the cell boundary. We conclude that Rho flares constitute a damage control mechanism that reinstates barrier function when tight junctions become locally compromised because of normally occurring changes in cell shape and tissue tension., (Copyright © 2019 Elsevier Inc. All rights reserved.)

DNA-protein crosslinks (DPCs) are bulky lesions that interfere with DNA metabolism and therefore threaten genomic integrity. Recent studies implicate the metalloprotease SPRTN in S phase removal of DPCs, but how SPRTN is targeted to DPCs during DNA replication is unknown. Using Xenopus egg extracts that recapitulate replication-coupled DPC proteolysis, we show that DPCs can be degraded by SPRTN or the proteasome, which act as independent DPC proteases. Proteasome recruitment requires DPC polyubiquitylation, which is partially dependent on the ubiquitin ligase activity of TRAIP. In contrast, SPRTN-mediated DPC degradation does not require DPC polyubiquitylation but instead depends on nascent strand extension to within a few nucleotides of the lesion, implying that polymerase stalling at the DPC activates SPRTN on both leading and lagging strand templates. Our results demonstrate that SPRTN and proteasome activities are coupled to DNA replication by distinct mechanisms that promote replication across immovable protein barriers., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Local translation is rapidly regulated by extrinsic signals during neural wiring, but its control mechanisms remain elusive. Here we show that the extracellular cue Sema3A induces an initial burst in local translation that precisely controls phosphorylation of the translation initiation factor eIF2α via the unfolded protein response (UPR) kinase PERK. Strikingly, in contrast to canonical UPR signaling, Sema3A-induced eIF2α phosphorylation bypasses global translational repression and underlies an increase in local translation through differential activity of eIF2B mediated by protein phosphatase 1. Ultrasensitive proteomics analysis of axons reveals 75 proteins translationally controlled via the Sema3A-p-eIF2α pathway. These include proteostasis- and actin cytoskeleton-related proteins but not canonical stress markers. Finally, we show that PERK signaling is needed for directional axon migration and visual pathway development in vivo. Thus, our findings reveal a noncanonical eIF2 signaling pathway that controls selective changes in axon translation and is required for neural wiring., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Early embryogenesis is accompanied by reductive cell divisions requiring that subcellular structures adapt to a range of cell sizes. The interphase nucleus and mitotic spindle scale with cell size through both physical and biochemical mechanisms, but control systems that coordinately scale intracellular structures are unknown. We show that the nuclear transport receptor importin α is modified by palmitoylation, which targets it to the plasma membrane and modulates its binding to nuclear localization signal (NLS)-containing proteins that regulate nuclear and spindle size in Xenopus egg extracts. Reconstitution of importin α targeting to the outer boundary of extract droplets mimicking cell-like compartments recapitulated scaling relationships observed during embryogenesis, which were altered by inhibitors that shift levels of importin α palmitoylation. Modulation of importin α palmitoylation in human cells similarly affected nuclear and spindle size. These experiments identify importin α as a conserved surface area-to-volume sensor that scales intracellular structures to cell size., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Covalent DNA-protein cross-links (DPCs) impede replication fork progression and threaten genome integrity. Using Xenopus egg extracts, we previously showed that replication fork collision with DPCs causes their proteolysis, followed by translesion DNA synthesis. We show here that when DPC proteolysis is blocked, the replicative DNA helicase CMG (CDC45, MCM2-7, GINS), which travels on the leading strand template, bypasses an intact leading strand DPC. Single-molecule imaging reveals that GINS does not dissociate from CMG during bypass and that CMG slows dramatically after bypass, likely due to uncoupling from the stalled leading strand. The DNA helicase RTEL1 facilitates bypass, apparently by generating single-stranded DNA beyond the DPC. The absence of RTEL1 impairs DPC proteolysis, suggesting that CMG must bypass the DPC to enable proteolysis. Our results suggest a mechanism that prevents inadvertent CMG destruction by DPC proteases, and they reveal CMG's remarkable capacity to overcome obstacles on its translocation strand., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Local translation regulates the axonal proteome, playing an important role in neuronal wiring and axon maintenance. How axonal mRNAs are localized to specific subcellular sites for translation, however, is not understood. Here we report that RNA granules associate with endosomes along the axons of retinal ganglion cells. RNA-bearing Rab7a late endosomes also associate with ribosomes, and real-time translation imaging reveals that they are sites of local protein synthesis. We show that RNA-bearing late endosomes often pause on mitochondria and that mRNAs encoding proteins for mitochondrial function are translated on Rab7a endosomes. Disruption of Rab7a function with Rab7a mutants, including those associated with Charcot-Marie-Tooth type 2B neuropathy, markedly decreases axonal protein synthesis, impairs mitochondrial function, and compromises axonal viability. Our findings thus reveal that late endosomes interact with RNA granules, translation machinery, and mitochondria and suggest that they serve as sites for regulating the supply of nascent pro-survival proteins in axons., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

DNA methylation and H3K9me are hallmarks of heterochromatin in plants and mammals, and are successfully maintained across generations. The biochemical and structural basis for this maintenance is poorly understood. The maintenance DNA methyltransferase from Zea mays, ZMET2, recognizes dimethylation of H3K9 via a chromodomain (CD) and a bromo adjacent homology (BAH) domain, which flank the catalytic domain. Here, we show that dinucleosomes are the preferred ZMET2 substrate, with DNA methylation preferentially targeted to linker DNA. Electron microscopy shows one ZMET2 molecule bridging two nucleosomes within a dinucleosome. We find that the CD stabilizes binding, whereas the BAH domain enables allosteric activation by the H3K9me mark. ZMET2 further couples recognition of H3K9me to an increase in the specificity for hemimethylated versus unmethylated DNA. We propose a model in which synergistic coupling between recognition of nucleosome spacing, H3K9 methylation, and DNA modification allows ZMET2 to maintain DNA methylation in heterochromatin with high fidelity., (Copyright © 2018. Published by Elsevier Inc.)

To couple the fidelity of patch-clamp recording with a more high-throughput screening capability, we pioneered a, to our knowledge, novel approach to single-channel recording that we named "optical patch clamp." By using highly sensitive fluorescent Ca 2+ indicator dyes in conjunction with total internal fluorescence microscopy techniques, we monitor Ca 2+ flux through individual Ca 2+ -permeable channels. This approach provides information about channel gating analogous to patch-clamp recording at a time resolution of ∼2 ms with the additional advantage of being massively parallel, providing simultaneous and independent recording from thousands of channels in the native environment. However, manual analysis of the data generated by this technique presents severe challenges because a video recording can include many thousands of frames. To overcome this bottleneck, we developed an image processing and analysis framework called CellSpecks capable of detecting and fully analyzing the kinetics of ion channels within a video sequence. By using randomly generated synthetic data, we tested the ability of CellSpecks to rapidly and efficiently detect and analyze the activity of thousands of ion channels, including openings for a few milliseconds. Here, we report the use of CellSpecks for the analysis of experimental data acquired by imaging muscle nicotinic acetylcholine receptors and the Alzheimer's disease-associated amyloid β pores with multiconductance levels in the plasma membrane of Xenopus laevis oocytes. We show that CellSpecks can accurately and efficiently generate location maps and create raw and processed fluorescence time traces; histograms of mean open times, mean close times, open probabilities, durations, and maximal amplitudes; and a "channel chip" showing the activity of all channels as a function of time. Although we specifically illustrate the application of CellSpecks for analyzing data from Ca 2+ channels, it can be easily customized to analyze other spatially and temporally localized signals., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

There is renewed interest in linker histone (LH)-nucleosome binding and how LHs influence eukaryotic DNA compaction. For a long time, the goal was to uncover "the structure of the chromatosome," but recent studies of LH-nucleosome complexes have revealed an ensemble of structures. Notably, the reconstituted LH-nucleosome complexes used in experiments rarely correspond to the sequence combinations present in organisms. For a full understanding of the determinants of the distribution of the chromatosome structural ensemble, studies must include a complete description of the sequences and experimental conditions used, and be designed to enable systematic evaluation of sequence and environmental effects., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The seven-transmembrane-spanning protein Smoothened is the central transducer in Hedgehog signaling, a pathway fundamental in development and in cancer. Smoothened is activated by cholesterol binding to its extracellular cysteine-rich domain (CRD). How this interaction leads to changes in the transmembrane domain and Smoothened activation is unknown. Here, we report crystal structures of sterol-activated Smoothened. The CRD undergoes a dramatic reorientation, allosterically causing the transmembrane domain to adopt a conformation similar to active G-protein-coupled receptors. We show that Smoothened contains a unique inhibitory π-cation lock, which is broken on activation and is disrupted in constitutively active oncogenic mutants. Smoothened activation opens a hydrophobic tunnel, suggesting a pathway for cholesterol movement from the inner membrane leaflet to the CRD. All Smoothened antagonists bind the transmembrane domain and block tunnel opening, but cyclopamine also binds the CRD, inducing the active transmembrane conformation. Together, these results define the mechanisms of Smoothened activation and inhibition., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Oocytes have a remarkable ability to reactivate silenced genes in somatic cells. However, it is not clear how the chromatin architecture of somatic cells affects this transcriptional reprogramming. Here, we investigated the relationship between the chromatin opening and transcriptional activation. We reveal changes in chromatin accessibility and their relevance to transcriptional reprogramming after transplantation of somatic nuclei into Xenopus oocytes. Genes that are silenced, but have pre-existing open transcription start sites in donor cells, are prone to be activated after nuclear transfer, suggesting that the chromatin signature of somatic nuclei influences transcriptional reprogramming. There are also activated genes associated with new open chromatin sites, and transcription factors in oocytes play an important role in transcriptional reprogramming from such genes. Finally, we show that genes resistant to reprogramming are associated with closed chromatin configurations. We conclude that chromatin accessibility is a central factor for successful transcriptional reprogramming in oocytes., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

DNA interstrand crosslinks (ICLs) are extremely cytotoxic, but the mechanism of their repair remains incompletely understood. Using Xenopus egg extracts, we previously showed that repair of a cisplatin ICL is triggered when two replication forks converge on the lesion. After CDC45/MCM2-7/GINS (CMG) ubiquitylation and unloading by the p97 segregase, FANCI-FANCD2 promotes DNA incisions by XPF-ERCC1, leading to ICL unhooking. Here, we report that, during this cell-free ICL repair reaction, one of the two converged forks undergoes reversal. Fork reversal fails when CMG unloading is inhibited, but it does not require FANCI-FANCD2. After one fork has undergone reversal, the opposing fork that still abuts the ICL undergoes incisions. Our data show that replication fork reversal at an ICL requires replisome disassembly. We present a revised model of ICL repair that involves a reversed fork intermediate., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mitosis is induced by the activation of the cyclin B/cdk1 feedback loop that creates a bistable state. The triggering factor promoting active cyclin B/cdk1 switch has been assigned to cyclin B/cdk1 accumulation during G2. However, this complex is rapidly inactivated by Wee1/Myt1-dependent phosphorylation of cdk1 making unlikely a triggering role of this kinase in mitotic commitment. Here we show that cyclin A/cdk1 kinase is the factor triggering mitosis. Cyclin A/cdk1 phosphorylates Bora to promote Aurora A-dependent Plk1 phosphorylation and activation and mitotic entry. We demonstrate that Bora phosphorylation by cyclin A/cdk1 is both necessary and sufficient for mitotic commitment. Finally, we identify a site in Bora whose phosphorylation by cyclin A/cdk1 is required for mitotic entry. We constructed a mathematical model confirming the essential role of this kinase in mitotic commitment. Overall, our results uncover the molecular mechanism by which cyclin A/cdk1 triggers mitotic entry., (Copyright © 2018 Elsevier Inc. All rights reserved.)

During the maternal-to-zygotic transition (MZT), maternal RNAs are actively degraded and replaced by newly synthesized zygotic transcripts in a highly coordinated manner. However, it remains largely unknown how maternal mRNA decay is triggered in early vertebrate embryos. Here, through genome-wide profiling of RNA abundance and 3' modification, we show that uridylation is induced at the onset of maternal mRNA clearance. The temporal control of uridylation is conserved in vertebrates. When the homologs of terminal uridylyltransferases TUT4 and TUT7 (TUT4/7) are depleted in zebrafish and Xenopus, maternal mRNA clearance is significantly delayed, leading to developmental defects during gastrulation. Short-tailed mRNAs are selectively uridylated by TUT4/7, with the highly uridylated transcripts degraded faster during the MZT than those with unmodified poly(A) tails. Our study demonstrates that uridylation plays a crucial role in timely mRNA degradation, thereby allowing the progression of early development., (Copyright © 2018 Elsevier Inc. All rights reserved.)

DNA replication in the embryo of Xenopus laevis changes dramatically at the mid-blastula transition (MBT), with Y RNA-independent random initiation switching to Y RNA-dependent initiation at specific origins. Here, we identify xNuRD, an MTA2-containing assemblage of the nucleosome remodeling and histone deacetylation complex NuRD, as an essential factor in pre-MBT Xenopus embryos that overcomes a functional requirement for Y RNAs during DNA replication. Human NuRD complexes have a different subunit composition than xNuRD and do not support Y RNA-independent initiation of DNA replication. Blocking or immunodepletion of xNuRD inhibits DNA replication initiation in isolated nuclei in vitro and causes inhibition of DNA synthesis, developmental delay, and embryonic lethality in early embryos. xNuRD activity declines after the MBT, coinciding with dissociation of the complex and emergence of Y RNA-dependent initiation. Our data thus reveal an essential role for a NuRD complex as a DNA replication factor during early Xenopus development., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cilia are organelles specialized for movement and signaling. To infer when during evolution signaling pathways became associated with cilia, we characterized the proteomes of cilia from sea urchins, sea anemones, and choanoflagellates. We identified 437 high-confidence ciliary candidate proteins conserved in mammals and discovered that Hedgehog and G-protein-coupled receptor pathways were linked to cilia before the origin of bilateria and transient receptor potential (TRP) channels before the origin of animals. We demonstrated that candidates not previously implicated in ciliary biology localized to cilia and further investigated ENKUR, a TRP channel-interacting protein identified in the cilia of all three organisms. ENKUR localizes to motile cilia and is required for patterning the left-right axis in vertebrates. Moreover, mutation of ENKUR causes situs inversus in humans. Thus, proteomic profiling of cilia from diverse eukaryotes defines a conserved ciliary proteome, reveals ancient connections to signaling, and uncovers a ciliary protein that underlies development and human disease., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The Na + ,K + -ATPase is a plasma membrane ion transporter of high physiological importance for ion homeostasis and cellular excitability in electrically active tissues. Mutations in the genes coding for Na + ,K + -ATPase α-subunit isoforms lead to severe human pathologies including Familial Hemiplegic Migraine type 2, Alternating Hemiplegia of Childhood, Rapid-onset Dystonia Parkinsonism, or epilepsy. Many of the reported mutations lead to change- or loss-of-function effects, whereas others do not alter the functional properties, but lead to, e.g., reduced protein stability, reduced protein expression, or defective plasma membrane targeting. Na + ,K + -ATPase frequently assembles with other membrane transporters or cellular matrix proteins in specialized plasma membrane microdomains, but the effects of these interactions on targeting or protein mobility are elusive so far. Mutation of established interaction motifs of the Na + ,K + -ATPase with ankyrin B and caveolin-1 are expected to result in changes in plasma membrane targeting, changes of the localization pattern, and of the diffusion behavior of the enzyme. We studied the consequences of mutations in these binding sites by monitoring diffusion of eGFP-labeled Na + ,K + -ATPase constructs in the plasma membrane of HEK293T cells by fluorescence correlation spectroscopy as well as fluorescence recovery after photobleaching or photoswitching, and observed significant differences compared to the wild-type enzyme, with synergistic effects for combinations of interaction site mutations. These measurements expand the possibilities to study the consequences of Na + ,K + -ATPase mutations and provide information about the interaction of Na + ,K + -ATPase α-isoforms with cellular matrix proteins, the cytoskeleton, or other membrane protein complexes., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Nucleosomes are believed to carry epigenetic information through the cell cycle, including through DNA replication. It has been known for decades that parental histones are reassembled on newly replicated chromatin, but the mechanisms underlying histone inheritance and dispersal during DNA replication are not fully understood. We monitored the fate of histones H3 or H4 from a single nucleosome through DNA replication in two in vitro systems. In the SV40 system, histones assembled on a single nucleosome positioning sequence can be inherited by their own daughter DNA but are dispersed from their original location. In Xenopus laevis extracts, histones are dynamic, and nucleosomes are repositioned independent of and prior to DNA replication. Nevertheless, a high fraction of histones H3 and H4 that are inherited through DNA replication remains near its starting location. Thus, inheritance of histone proteins and their dispersal can be mechanistically uncoupled., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Chromatin remodelers catalyze dynamic packaging of the genome by carrying out nucleosome assembly/disassembly, histone exchange, and nucleosome repositioning. Remodeling results in evenly spaced nucleosomes, which requires probing both sides of the nucleosome, yet the way remodelers organize sliding activity to achieve this task is not understood. Here, we show that the monomeric Chd1 remodeler shifts DNA back and forth by dynamically alternating between different segments of the nucleosome. During sliding, Chd1 generates unstable remodeling intermediates that spontaneously relax to a pre-remodeled position. We demonstrate that nucleosome sliding is tightly controlled by two regulatory domains: the DNA-binding domain, which interferes with sliding when its range is limited by a truncated linking segment, and the chromodomains, which play a key role in substrate discrimination. We propose that active interplay of the ATPase motor with the regulatory domains may promote dynamic nucleosome structures uniquely suited for histone exchange and chromatin reorganization during transcription., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The early Xenopus laevis embryo is replete with dynamic spatial waves. One such wave, the cell division wave, emerges from the collective cell division timing of first tens and later hundreds of cells throughout the embryo. Here, we show that cell division waves do not propagate between neighboring cells and do not rely on cell-to-cell coupling to maintain their division timing. Instead, intrinsic variation in division period autonomously and gradually builds these striking patterns of cell division. Disrupting this pattern of division by placing embryos in a temperature gradient resulted in highly asynchronous entry to the midblastula transition and misexpression of the mesodermal marker Xbra. Remarkably, this gene expression defect is corrected during involution, resulting in delayed yet normal Xbra expression and viable embryos. This implies the existence of a previously unknown mechanism for normalizing mesodermal gene expression during involution., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Brca2 deficiency causes Mre11-dependent degradation of nascent DNA at stalled forks, leading to cell lethality. To understand the molecular mechanisms underlying this process, we isolated Xenopus laevis Brca2. We demonstrated that Brca2 protein prevents single-stranded DNA gap accumulation at replication fork junctions and behind them by promoting Rad51 binding to replicating DNA. Without Brca2, forks with persistent gaps are converted by Smarcal1 into reversed forks, triggering extensive Mre11-dependent nascent DNA degradation. Stable Rad51 nucleofilaments, but not RPA or Rad51 T131P mutant proteins, directly prevent Mre11-dependent DNA degradation. Mre11 inhibition instead promotes reversed fork accumulation in the absence of Brca2. Rad51 directly interacts with the Pol α N-terminal domain, promoting Pol α and δ binding to stalled replication forks. This interaction likely promotes replication fork restart and gap avoidance. These results indicate that Brca2 and Rad51 prevent formation of abnormal DNA replication intermediates, whose processing by Smarcal1 and Mre11 predisposes to genome instability., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Vertebrate centromeres are epigenetically defined by nucleosomes containing the histone H3 variant, CENP-A. CENP-A nucleosome assembly requires the three-protein Mis18 complex (Mis18α, Mis18β, and M18BP1) that recruits the CENP-A chaperone HJURP to centromeres, but how the Mis18 complex recognizes centromeric chromatin is unknown. Using Xenopus egg extract, we show that direct, cell-cycle-regulated binding of M18BP1 to CENP-A nucleosomes recruits the Mis18 complex to interphase centromeres to promote new CENP-A nucleosome assembly. We demonstrate that Xenopus M18BP1 binds CENP-A nucleosomes using a motif that is widely conserved except in mammals. The M18BP1 motif resembles a CENP-A nucleosome binding motif in CENP-C, and we show that CENP-C competes with M18BP1 for CENP-A nucleosome binding at centromeres. We show that both CENP-C and M18BP1 recruit HJURP to centromeres for new CENP-A assembly. This study defines cellular mechanisms for recruiting CENP-A assembly factors to existing CENP-A nucleosomes for the epigenetic inheritance of centromeres., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The early cell divisions of many metazoan embryos are rapid and occur in the near absence of transcription. At the mid-blastula transition (MBT), the cell cycle elongates and several processes become established including the onset of bulk transcription and cell-cycle checkpoints. How these events are timed and coordinated is poorly understood. Here we show in Xenopus laevis that developmental activation of the checkpoint kinase Chk1 at the MBT results in the SCF β-TRCP -dependent degradation of a limiting replication initiation factor Drf1. Inhibition of Drf1 is the primary mechanism by which Chk1 blocks cell-cycle progression in the early embryo and is an essential function of Chk1 at the blastula-to-gastrula stage of development. This study defines the downregulation of Drf1 as an important mechanism to coordinate the lengthening of the cell cycle and subsequent developmental processes., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Linker histones associate with nucleosomes to promote the formation of higher-order chromatin structure, but the underlying molecular details are unclear. We investigated the structure of a 197 bp nucleosome bearing symmetric 25 bp linker DNA arms in complex with vertebrate linker histone H1. We determined electron cryo-microscopy (cryo-EM) and crystal structures of unbound and H1-bound nucleosomes and validated these structures by site-directed protein cross-linking and hydroxyl radical footprinting experiments. Histone H1 shifts the conformational landscape of the nucleosome by drawing the two linkers together and reducing their flexibility. The H1 C-terminal domain (CTD) localizes primarily to a single linker, while the H1 globular domain contacts the nucleosome dyad and both linkers, associating more closely with the CTD-distal linker. These findings reveal that H1 imparts a strong degree of asymmetry to the nucleosome, which is likely to influence the assembly and architecture of higher-order structures., (Copyright © 2017 Elsevier Inc. All rights reserved.)

During brain wiring, cue-induced axon behaviors such as directional steering and branching are aided by localized mRNA translation. Different guidance cues elicit translation of subsets of mRNAs that differentially regulate the cytoskeleton, yet little is understood about how specific mRNAs are selected for translation. MicroRNAs (miRNAs) are critical translational regulators that act through a sequence-specific mechanism. Here, we investigate the local role of miRNAs in mRNA-specific translation during pathfinding of Xenopus laevis retinal ganglion cell (RGC) axons. Among a rich repertoire of axonal miRNAs, miR-182 is identified as the most abundant. Loss of miR-182 causes RGC axon targeting defects in vivo and impairs Slit2-induced growth cone (GC) repulsion. We find that miR-182 targets cofilin-1 mRNA, silencing its translation, and Slit2 rapidly relieves the repression without causing miR-182 degradation. Our data support a model whereby miR-182 reversibly gates the selection of transcripts for fast translation depending on the extrinsic cue., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

The mouth arises from the extreme anterior domain (EAD), a region where the ectoderm and endoderm are directly juxtaposed. Here, we identify a "pre-mouth array" in Xenopus that forms soon after the cranial neural crest has migrated to lie on either side of the EAD. Initially, EAD ectoderm comprises a wide and short epithelial mass that becomes narrow and tall with cells and nuclei changing shape, a characteristic of convergent extension. The resulting two rows of cells-the pre-mouth array-later split down the midline to surround the mouth opening. Neural crest is essential for convergent extension and likely signals to the EAD through the Wnt/planar cell polarity (PCP) pathway. Fzl7 receptor is locally required in EAD ectoderm, while Wnt11 ligand is required more globally. Indeed, heterologous cells expressing Wnt11 can elicit EAD convergent extension. The study reveals a precise cellular mechanism that positions and contributes to the future mouth., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Many plasma membrane channels form oligomeric assemblies, and heterooligomerization has been described as a distinctive feature of some protein families. In the particular case of plant plasma membrane aquaporins (PIPs), PIP1 and PIP2 monomers interact to form heterotetramers. However, the biological properties of the different heterotetrameric configurations formed by PIP1 and PIP2 subunits have not been addressed yet. Upon coexpression of tandem PIP2-PIP1 dimers in Xenopus oocytes, we can address, for the first time to our knowledge, the functional properties of single heterotetrameric species having 2:2 stoichiometry. We have also coexpressed PIP2-PIP1 dimers with PIP1 and PIP2 monomers to experimentally investigate the localization and biological activity of each tetrameric assembly. Our results show that PIP2-PIP1 heterotetramers can assemble with 3:1, 1:3, or 2:2 stoichiometry, depending on PIP1 and PIP2 relative expression in the cell. All PIP2-PIP1 heterotetrameric species localize at the plasma membrane and present the same water transport capacity. Furthermore, the contribution of any heterotetrameric assembly to the total water transport through the plasma membrane doubles the contribution of PIP2 homotetramers. Our results also indicate that plasma membrane water transport can be modulated by the coexistence of different tetrameric species and by intracellular pH. Moreover, all the tetrameric species present similar cooperativity behavior for proton sensing. These findings throw light on the functional properties of PIP tetramers, showing that they have flexible stoichiometry dependent on the quantity of PIP1 and PIP2 molecules available. This represents, to our knowledge, a novel regulatory mechanism to adjust water transport across the plasma membrane., (Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

While components of the pathway that establishes left-right asymmetry have been identified in diverse animals, from vertebrates to flies, it is striking that the genes involved in the first symmetry-breaking step remain wholly unknown in the most obviously chiral animals, the gastropod snails. Previously, research on snails was used to show that left-right signaling of Nodal, downstream of symmetry breaking, may be an ancestral feature of the Bilateria [1 and 2]. Here, we report that a disabling mutation in one copy of a tandemly duplicated, diaphanous-related formin is perfectly associated with symmetry breaking in the pond snail. This is supported by the observation that an anti-formin drug treatment converts dextral snail embryos to a sinistral phenocopy, and in frogs, drug inhibition or overexpression by microinjection of formin has a chirality-randomizing effect in early (pre-cilia) embryos. Contrary to expectations based on existing models [3, 4 and 5], we discovered asymmetric gene expression in 2- and 4-cell snail embryos, preceding morphological asymmetry. As the formin-actin filament has been shown to be part of an asymmetry-breaking switch in vitro [6 and 7], together these results are consistent with the view that animals with diverse body plans may derive their asymmetries from the same intracellular chiral elements [8]., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cilia are microtubule-based projections that function in the movement of extracellular fluid. This requires cilia to be: (1) motile and driven by dynein complexes and (2) correctly polarized on the surface of cells, which requires planar cell polarity (PCP). Few factors that regulate both processes have been discovered. We reveal that C21orf59/Kurly (Kur), a cytoplasmic protein with some enrichment at the base of cilia, is needed for motility; zebrafish mutants exhibit characteristic developmental abnormalities and dynein arm defects. kur was also required for proper cilia polarization in the zebrafish kidney and the larval skin of Xenopus laevis. CRISPR/Cas9 coupled with homologous recombination to disrupt the endogenous kur locus in Xenopus resulted in the asymmetric localization of the PCP protein Prickle2 being lost in mutant multiciliated cells. Kur also makes interactions with other PCP components, including Disheveled. This supports a model wherein Kur plays a dual role in cilia motility and polarization., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Neurulation is a critical period in all vertebrates and results in the formation of the neural tube, which gives rise to the CNS. Apical constriction is one of the fundamental morphogenetic movements that drives neural tube closure. Using live imaging, we show that apical constriction during the neurulation is a stepwise process driven by cell-autonomous and asynchronous contraction pulses followed by stabilization steps. Our data suggest that contraction events are triggered by cell-autonomous Ca(2+) flashes and are driven by a transient contractile apical pool of actin. In addition, we provide evidence that the cell autonomy and asynchrony of contraction are required for the correct spatial distribution of constriction and, as a result, are critical for tissue morphogenesis. Finally, we identify Calpain2 as a regulator of apical constriction and show that it is required for the stabilization step, but is dispensable during contraction., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

A biochemical explanation of development from the fertilized egg to the adult requires an understanding of the proteins and RNAs expressed over time during embryogenesis. We present a comprehensive characterization of protein and mRNA dynamics across early development in Xenopus. Surprisingly, we find that most protein levels change little and duplicated genes are expressed similarly. While the correlation between protein and mRNA levels is poor, a mass action kinetics model parameterized using protein synthesis and degradation rates regresses protein dynamics to RNA dynamics, corrected for initial protein concentration. This study provides detailed data for absolute levels of ∼10,000 proteins and ∼28,000 transcripts via a convenient web portal, a rich resource for developmental biologists. It underscores the lasting impact of maternal dowry, finds surprisingly few cases where degradation alone drives a change in protein level, and highlights the importance of transcription in shaping the dynamics of the embryonic proteome., (Copyright © 2015 Elsevier Inc. All rights reserved.)