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Nuclear pore complex (NPC) mediates nucleocytoplasmic shuttling. Here we present single-particle cryo-electron microscopy structure of the inner ring (IR) subunit from the Xenopus laevis NPC at an average resolution of 4.2 Å. A homo-dimer of Nup205 resides at the center of the IR subunit, flanked by two molecules of Nup188. Four molecules of Nup93 each places an extended helix into the axial groove of Nup205 or Nup188, together constituting the central scaffold. The channel nucleoporin hetero-trimer of Nup62/58/54 is anchored on the central scaffold. Six Nup155 molecules interact with the central scaffold and together with the NDC1-ALADIN hetero-dimers anchor the IR subunit to the nuclear envelope and to outer rings. The scarce inter-subunit contacts may allow sufficient latitude in conformation and diameter of the IR. Our structure reveals the molecular basis for the IR subunit assembly of a vertebrate NPC., (© 2022. The Author(s).)

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design efficient remedies for diseases. Technologies enabling accurate identification and quantification of diverse types and large numbers of proteins would provide still missing information on molecular mechanisms orchestrating tissue and organism development in space and time. Here, we present a mass spectrometry-based protocol that enables the measurement of thousands of proteins in identified cell lineages in Xenopus laevis (frog) embryos. The approach builds on reproducible cell-fate maps and established methods to identify, fluorescently label, track, and sample cells and their progeny (clones) from this model of vertebrate development. After collecting cellular contents using microsampling or isolating cells by dissection or fluorescence-activated cell sorting, proteins are extracted and processed for bottom-up proteomic analysis. Liquid chromatography and capillary electrophoresis are used to provide scalable separation for protein detection and quantification with high-resolution mass spectrometry (HRMS). Representative examples are provided for the proteomic characterization of neural-tissue fated cells. Cell-lineage-guided HRMS proteomics is adaptable to different tissues and organisms. It is sufficiently sensitive, specific, and quantitative to peer into the spatio-temporal dynamics of the proteome during vertebrate development.

Xenopus laevis, the African clawed frog, undergoes seasonal estivation to survive periods of drought when its lake-bed habitats dry up. The frog can lose ∼30% of its total body water, leading to conditions of impaired blood flow and ischemia which risk cellular survival under these harsh conditions. MicroRNAs are short, noncoding, single-stranded RNAs 21-24 nt long that have been widely implicated in hypometabolic responses, and serve functions including apoptosis survival. The levels of three pro-apoptotic and four anti-apoptotic miRNAs were measured in liver and skeletal muscle of estivating X. laevis, and bioinformatic analysis was performed to verify potential mRNA targets of these miRNAs. Members of pro-apoptotic miRNAs miR-15a, miR-16, and miR-101 showed upregulation as a result of dehydration stress, while anti-apoptotic miRNAs miR-19b, miR-21, miR-92a, and miR-155 showed differential regulation between the two tissues. Together, these miRNAs act in a more diverse fashion than arbitrarily pro- or anti-apoptotic, and encompass functions ranging from the inhibition of cell proliferation through cell cycle arrest to the prevention of skeletal muscle atrophy., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: The Xenopus retinotectal circuit is organized topographically, where the dorsal-ventral axis of the retina maps respectively on to the ventral-dorsal axis of the tectum; axons from the nasal-temporal axis of the retina project respectively to the caudal-rostral axis of the tectum. Studies throughout the last two decades have shown that mechanisms involving molecular recognition of proper termination domains are at work guiding topographic organization. Such studies have shown that graded distribution of molecular cues is important for topographic mapping. However, the complement of molecular cues organizing topography along the developing optic nerve, and as retinal axons cross the chiasm and navigate towards and innervate their target in the tectum, remains unknown. Down syndrome cell adhesion molecule (DSCAM) has been characterized as a key molecule in axon guidance, making it a strong candidate involved in the topographic organization of retinal fibers along the optic path and at their target., Methods: Using a combination of whole-brain clearing and immunohistochemistry staining techniques we characterized DSCAM expression and the projection of ventral and dorsal retinal fibers starting from the eye, following to the optic nerve and chiasm, and into the terminal target in the optic tectum in Xenopus laevis tadpoles. We then assessed the effects of DSCAM on the establishment of retinotopic maps through spatially and temporally targeted DSCAM knockdown on retinal ganglion cells (RGCs) with axons innervating the optic tectum., Results: Highest expression of DSCAM was localized to the ventral posterior region of the optic nerve and chiasm; this expression pattern coincides with ventral fibers derived from ventral RGCs. Targeted downregulation of DSCAM expression on ventral RGCs affected the segregation of medial axon fibers from their dorsal counterparts within the tectal neuropil, indicating that DSCAM plays a role in retinotopic organization., Conclusion: These findings together with previous studies demonstrating cell-autonomous roles for DSCAM during the development of pre- and postsynaptic arbors in the Xenopus retinotectal circuit indicates that DSCAM exerts multiple roles in coordinating axon targeting and structural connectivity in the developing vertebrate visual system., (© 2022. The Author(s).)

Nuclear pore complex (NPC) shuttles cargo across the nuclear envelope. Here we present single-particle cryo-EM structure of the nuclear ring (NR) subunit from Xenopus laevis NPC at an average resolution of 5.6 Å. The NR subunit comprises two 10-membered Y complexes, each with the nucleoporin ELYS closely associating with Nup160 and Nup37 of the long arm. Unlike the cytoplasmic ring (CR) or inner ring (IR), the NR subunit contains only one molecule each of Nup205 and Nup93. Nup205 binds both arms of the Y complexes and interacts with the stem of inner Y complex from the neighboring subunit. Nup93 connects the stems of inner and outer Y complexes within the same NR subunit, and places its N-terminal extended helix into the axial groove of Nup205 from the neighboring subunit. Together with other structural information, we have generated a composite atomic model of the central ring scaffold that includes the NR, IR, and CR. The IR is connected to the two outer rings mainly through Nup155. This model facilitates functional understanding of vertebrate NPC., (© 2022. The Author(s).)

It has previously been reported that in ex vivo planar explants prepared from Xenopus laevis embryos, the intracellular pH (pHi) increases in cells of the dorsal ectoderm from stage 10.5 to 11.5 (i.e. 11-12.5 hpf). It was proposed that such increases (potentially due to H+ being extruded, sequestered, or buffered in some manner), play a role in regulating neural induction. Here, we used an extracellular ion-selective electrode to non-invasively measure H+ fluxes at eight locations around the equatorial circumference of intact X. laevis embryos between stages 9-12 (˜7-13.25 hpf). We showed that at stages 9-11, there was a small H+ efflux recorded from all the measuring positions. At stage 12 there was a small, but significant, increase in the efflux of H+ from most locations, but the efflux from the dorsal side of the embryo was significantly greater than from the other positions. Embryos were also treated from stages 9-12 with bafilomycin A1, to block the activity of the ATP-driven H+ pump. By stage 22 (24 hpf), these embryos displayed retarded development, arresting before the end of gastrulation and therefore did not display the usual anterior and neural structures, which were observed in the solvent-control embryos. In addition, expression of the early neural gene, Zic3, was absent in treated embryos compared with the solvent controls. Together, our new in vivo data corroborated and extended the earlier explant-derived report describing changes in pHi that were suggested to play a role during neural induction in X. laevis embryos.

Condensins compact chromosomes to promote their equal segregation during mitosis, but the mechanism of condensin engagement with and action on chromatin is incompletely understood. Here, we show that the general transcription factor TFIIH complex is continuously required to establish and maintain a compacted chromosome structure in transcriptionally silent Xenopus egg extracts. Inhibiting the DNA-dependent ATPase activity of the TFIIH complex subunit XPB rapidly and reversibly induces a complete loss of chromosome structure and prevents the enrichment of condensins I and II, but not topoisomerase II, on chromatin. In addition, inhibiting TFIIH prevents condensation of both mouse and Xenopus nuclei in Xenopus egg extracts, which suggests an evolutionarily conserved mechanism of TFIIH action. Reducing nucleosome density through partial histone depletion restores chromosome structure and condensin enrichment in the absence of TFIIH activity. We propose that the TFIIH complex promotes mitotic chromosome condensation by dynamically altering the chromatin environment to facilitate condensin loading and condensin-dependent loop extrusion., Competing Interests: JH, RC, WP, MB, LJ, AK No competing interests declared

Failures of neural tube closure are common and serious birth defects, yet we have a poor understanding of the interaction of genetics and cell biology during neural tube closure. Additionally, mutations that cause neural tube defects (NTDs) tend to affect anterior or posterior regions of the neural tube but rarely both, indicating a regional specificity to NTD genetics. To better understand the regional specificity of cell behaviors during neural tube closure, we analyzed the dynamic localization of actin and N-cadherin via high-resolution tissue-level time-lapse microscopy during Xenopus neural tube closure. To investigate the regionality of gene function, we generated mosaic mutations in shroom3 , a key regulator or neural tube closure. This new analytical approach elucidates several differences between cell behaviors during cranial/anterior and spinal/posterior neural tube closure, provides mechanistic insight into the function of shroom3, and demonstrates the ability of tissue-level imaging and analysis to generate cell biological mechanistic insights into neural tube closure., Competing Interests: AB, JK, HS, JW No competing interests declared, (© 2022, Baldwin et al.)

Background: Development of paired sensory organs is a highly complex and coordinated process. These organs arise from ectodermal thickenings in the cephalic region known as cranial placodes. We have previously shown that Zic1 is a critical regulator for the formation of the pre-placodal region (PPR), the common territory for the development of all cranial placodes in Xenopus laevis., Results: In this study, we have analyzed a number of Zic1 targets for their expression during PPR patterning, as well as their regulation by retinoic acid (RA) and one of its major metabolites, 4-oxo-RA. Our findings show that anteriorly Zic1 regulates several transcription factors, Crx, Fezf2, Nkx3-1, and Xanf1 as well as a serine/threonine/tyrosine kinase, Pkdcc.2. These factors are all expressed in the vicinity of the PPR and as such are candidate regulators of placode formation downstream of Zic1. In addition to their differential regulation by RA, we find that 4-oxo-RA is also capable of modulating the expression of these genes, as well as a broad array of RA-regulated genes., Conclusion: Our data highlight the complexity of retinoid-mediated regulation required for Zic1-activated anterior structure specification in Xenopus, and the potential physiological role of 4-oxo-RA in cranial placode development., (© 2021 American Association for Anatomy.)

The Ventx family is one of the subfamilies of the ANTP ( antennapedia ) superfamily and belongs to the NK-like (NKL) subclass. Ventx is a homeobox transcription factor and has a DNA-interacting domain that is evolutionarily conserved throughout vertebrates. It has been extensively studied in Xenopus , zebrafish, and humans. The Ventx family contains transcriptional repressors widely involved in embryonic development and tumorigenesis in vertebrates. Several studies have documented that the Ventx family inhibited dorsal mesodermal formation, neural induction, and head formation in Xenopus and zebrafish. Moreover, Ventx2.2 showed functional similarities to Nanog and Barx1, leading to pluripotency and neural-crest migration in vertebrates. Among them, Ventx protein is an orthologue of the Ventx family in humans. Studies have demonstrated that human Ventx was strongly associated with myeloid-cell differentiation and acute myeloid leukemia. The therapeutic potential of Ventx family inhibition in combating cancer progression in humans is discussed. Additionally, we briefly discuss genome evolution, gene duplication, pseudo-allotetraploidy, and the homeobox family in Xenopus .

Cations such as Cs + and Ba 2+ are known to block K + currents by entering an open channel and binding to the selectivity filter, where they obstruct the pore and block diffusion of the permeant ion. This obstruction is voltage- and K + -dependent and is relieved by the trans permeant ion flux. The present patch-clamp study on Xenopus muscle cells shows that, unlike the voltage-activated K + (Kv) channels, blockade of the inward rectifier K + (Kir) channels by external foreign cations results from the combination of pore obstruction with a new and independent mechanism. This new blockade is independent of the K + concentrations and flux and acts indiscriminately on both the outward and the inward Kir components. External Cs + and Ba 2+ compete for this blockade with free access to common channel sites. These features suggest that the blocking cations do not need to enter the channel for this new mechanism, and should bind to the extracellular side of the channel. When K + fluxes are flowing outward, the pore obstruction is relieved for both Kir and Kv currents, and the K + -independent blockade here described is responsible for a selective Kir inhibition, justifying the use of these external cations as tools in cell physiology studies., (© 2022 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Insect nicotinic acetylcholine receptors (nAChRs) are molecular targets of highly effective insecticides such as neonicotinoids. Functional expression of these receptors provides useful insights into their functional and pharmacological properties. Here, we report that the α5 nAChR subunit of the honey bee, Apis mellifera, functionally expresses in Xenopus laevis oocytes, which is the first time a homomeric insect nAChR has been robustly expressed in a heterologous system without the need for chaperone proteins. Using two-electrode voltage-clamp electrophysiology we show that the α5 receptor has low sensitivity to acetylcholine with an EC 50 of 2.37 mM. However, serotonin acts as an agonist with a considerably lower EC 50 at 119 μM that is also more efficacious than acetylcholine in activating the receptor. Molecular modelling indicates that residues in the complementary binding site may be involved in the selectivity towards serotonin. This is the first report of a ligand-gated ion channel activated by serotonin from an insect and phylogenetic analysis shows that the α5 subunit of A. mellifera and other non-Dipteran insects, including pest species, belong to a distinct subgroup of subunits, which may represent targets for the development of novel classes of insecticides., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Proper function of the body is maintained by an intricate interaction and communication among cells. during the animal development how these cells are formed and maintained is an important yet elusive. Understanding of how cells such as muscle and nerve cells maintain their identities would enable us to control the diseases which include malfunctioning in cellular identities such as cancer. In this article, we describe how the concept of formation and maintenance of cell identities has changed over the last 100 years. We will also briefly describe our current experimental work which includes transcriptional dynamics, and protein-protein interaction and how they are bringing new molecular insights. We also describe liquid-liquid phase separation as a potential new mechanism for the stability of gene expression in the non dvididng specialised cells of Xenopus oocytes., (Copyright © 2021. Published by Elsevier Inc.)

Retinitis pigmentosa is an inherited retinal dystrophy that ultimately leads to blindness due to the progressive degeneration of rod photoreceptors and the subsequent non-cell autonomous death of cones. Rhodopsin is the most frequently mutated gene in this disease. We here developed rhodopsin gene editing-based models of retinitis pigmentosa in two Xenopus species, Xenopus laevis and Xenopus tropicalis , by using CRISPR/Cas9 technology. In both of them, loss of rhodopsin function results in massive rod cell degeneration characterized by progressive shortening of outer segments and occasional cell death. This is followed by cone morphology deterioration. Despite these apparently similar degenerative environments, we found that Müller glial cells behave differently in Xenopus laevis and Xenopus tropicalis . While a significant proportion of Müller cells re-enter into the cell cycle in Xenopus laevis , their proliferation remains extremely limited in Xenopus tropicalis . This work thus reveals divergent responses to retinal injury in closely related species. These models should help in the future to deepen our understanding of the mechanisms that have shaped regeneration during evolution, with tremendous differences across vertebrates.

We report both the design of a high-throughput MICROFASP (a miniaturized filter aided sample preparation) system and its use for the comprehensive proteomic analysis of single blastomeres isolated from 50-cell stage Xenopus laevis embryos (∼200 ng of yolk-free protein/blastomere). A single run of the MICROFASP system was used to process 146 of these blastomeres in parallel. Three samples failed to generate signals presumably due to membrane clogging. Two cells were lost due to operator error. Of the surviving samples, 32 were analyzed using a Q Exactive HF mass spectrometer in survey experiments (data not included). The 109 remaining blastomeres were analyzed using a capillary LC-ESI-MS/MS system coupled to an Orbitrap Fusion Lumos mass spectrometer, which identified a total of 4189 protein groups and 40,998 unique peptides. On average, 3468 ± 229 protein groups and 14,525 ± 2437 unique peptides were identified from each blastomere, which is the highest throughput and deepest proteome coverage to date of single blastomeres at this stage of development. We also compared two dissociation buffers, Newport and calcium-magnesium-free (CMFM) buffers; the two buffers generated similar numbers of protein identifications (3615 total protein IDs from use of the Newport dissociation buffer and 3671 total protein IDs from use of the CMFM buffer).

Xenopus laevis tadpoles possess high regenerative ability and can regenerate functional tails after amputation. An early event in regeneration is the induction of undifferentiated cells that form the regenerated tail. We previously reported that interleukin-11 (il11) is upregulated immediately after tail amputation to induce undifferentiated cells of different cell lineages, indicating a key role of il11 in initiating tail regeneration. As Il11 is a secretory factor, Il11 receptor-expressing cells are thought to mediate its function. X. laevis has a gene annotated as interleukin 11 receptor subunit alpha on chromosome 1L (il11ra.L), a putative subunit of the Il11 receptor complex, but its function has not been investigated. Here, we show that nuclear localization of phosphorylated Stat3 induced by Il11 is abolished in il11ra.L knocked-out culture cells, strongly suggesting that il11ra.L encodes an Il11 receptor component. Moreover, knockdown of il11ra.L impaired tadpole tail regeneration, suggesting its indispensable role in tail regeneration. We also provide a model showing that Il11 functions via il11ra.L-expressing cells in a non-cell autonomous manner. These results highlight the importance of il11ra.L-expressing cells in tail regeneration., (© 2022. The Author(s).)

Wnt signalling plays an eminent role in development, stem cell growth, and tissue homeostasis. Much of what we know about Wnt signalling, we owe to research in developmental biology. Here I review some salient discoveries in the older literature, beginning with the Lithium experiments in sea urchin by Curt Herbst in the 1890ies, when unknown to him he observed the gradual effects of Wnt overactivation upon embryonic axis formation. After revisiting key discoveries into Wingless signalling in Drosophila, I examine the role that the Xenopus embryo has played as model system in this regard. Not only were components of the Wnt cascade dissected and secreted Wnt antagonists discovered in Xenopus, but it also played a key role in unveiling the evolutionary conserved role of Wnt signalling in primary body axis formation. I conclude that Xenopus developmental biology has played a major role in elucidating the mechanisms of embryonic Wnt signalling., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Shroom is a family of related proteins linked to the actin cytoskeleton, and one of them, xShroom1, is constitutively expressed in Xenopus laevis oocytes which is required for the expression of the epithelial sodium channel (ENaC). On the other hand, ENaC and the cystic fibrosis transmembrane regulator (CFTR) are co-expressed in many types of cells with a negative or positive interaction depending on the studied tissues. Here, we measured the amiloride-sensitive ENaC currents (INa amil ) and CFTR currents (I CFTR ) with voltage clamp techniques in oocytes co-injected with ENaC and/or CFTR and xShroom1 antisense oligonucleotides. The objective was to study the mechanism of regulation of ENaC by CFTR when xShroom1 was suppressed and the endocytic traffic of CFTR was blocked. CFTR activation had a measurable negative effect on ENaC and this activation resulted in a greater inhibition of INa amil than with xShroom1 antisense alone. Our results with Dynasore, a drug that acts as an inhibitor of endocytic pathways, suggest that the changes in INa amil by xShroom1 downregulation were probably due to an increment in channel endocytosis. An opposite effect was observed when I CFTR was measured. Thus, when xShroom1 was downregulated, the I CFTR was larger than in the control experiments and this effect is not observed with Dynasore. A speculative explanation could be that xShroom1 exerts a dual effect on the endocytic traffic of ENaC and CFTR and these actions were canceled with Dynasore. In the presence of Dynasore, no difference in either INa amil or I CFTR was observed when xShroom1 was downregulated., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Here, we present a solid-supported membrane (SSM)-based electrophysiological approach to study sugar binding and Na + /glucose cotransport by SGLT1 in membrane vesicles. SSM-based electrophysiology delivers a cumulative real-time current readout from numerous SGLT1 proteins simultaneously using a gold-coated sensor chip. In contrast to conventional techniques, which mainly operate with voltage steps, currents are triggered by sugar or sodium addition. Sugar concentration jumps in the presence of sodium lead to transport currents between 5 and 10 nA. Remarkably, in the absence of sodium (i.e. no transport), we observed fast pre-steady-state (PSS) currents with time constants between 3 and 10 ms. These PSS currents mainly originate from sugar binding. Sodium binding does not induce PSS currents. Due to high time resolution, PSS currents were distinguished from transport and eventually correlated with conformational transitions within the sugar translocation pathway. In addition, we analyzed the impact of driving forces on transport and binding currents, showing that membrane voltage and sodium concentration gradients lead to an increased transport rate without affecting sugar binding kinetics. We also compared Na + /sugar efflux with physiologically relevant influx and found similar transport rates, but lower affinity in efflux mode. SSM-based electrophysiology is a powerful technique, which overcomes bottlenecks for transport measurements observed in other techniques such as the requirement of labels or the lack of real-time data. Rapid solution exchange enables the observation of substrate-induced electrogenic events like conformational transitions, opening novel perspectives for in-depth functional studies of SGLT1 and other transporters., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Proteolytic activation of the epithelial sodium channel (ENaC) by aberrantly filtered serine proteases is thought to contribute to renal sodium retention in nephrotic syndrome. However, the identity of the responsible proteases remains elusive. This study evaluated factor VII activating protease (FSAP) as a candidate in this context. We analyzed FSAP in the urine of patients with nephrotic syndrome and nephrotic mice and investigated its ability to activate human ENaC expressed in Xenopus laevis oocytes. Moreover, we studied sodium retention in FSAP-deficient mice (Habp2 -/- ) with experimental nephrotic syndrome induced by doxorubicin. In urine samples from nephrotic humans, high concentrations of FSAP were detected both as zymogen and in its active state. Recombinant serine protease domain of FSAP stimulated ENaC-mediated whole-cell currents in a time- and concentration-dependent manner. Mutating the putative prostasin cleavage site in γ-ENaC (γRKRK178AAAA) prevented channel stimulation by the serine protease domain of FSAP. In a mouse model for nephrotic syndrome, active FSAP was present in nephrotic urine of Habp2 +/+ but not of Habp2 -/- mice. However, Habp2 -/- mice were not protected from sodium retention compared to nephrotic Habp2 +/+ mice. Western blot analysis revealed that in nephrotic Habp2 -/- mice, proteolytic cleavage of α- and γ-ENaC was similar to that in nephrotic Habp2 +/+ animals. In conclusion, active FSAP is excreted in the urine of nephrotic patients and mice and activates ENaC in vitro involving the putative prostasin cleavage site of γ-ENaC. However, endogenous FSAP is not essential for sodium retention in nephrotic mice., (© 2021. The Author(s).)

Previous studies show that the main cannabinoid receptor in the brain-cannabinoid type 1 receptor (CB1R)-is required for establishment of axonal projections in developing neurons but questions remain regarding the cellular and molecular mechanisms, especially in neurons developing in their native environment. We assessed the effects of CB1R signalling on growth cone filopodia and axonal projections of retinal ganglion cells (RGCs) in whole mount brains from Xenopus laevis tadpoles. Our results indicate that growth cones of RGC axons in brains from tadpoles exposed to a CB1R agonist had fewer filopodial protrusions, whereas growth cones from tadpoles exposed to a CB1R inverse agonist had more filopodia than growth cones of RGC axons in whole brains from control tadpoles. However, application of both the CB1R agonist and inverse agonist resulted in RGC axons that were overly dispersed and undulatory in the optic tract in situ. In addition, expression of a mutant for cadherin adhesive factor, β-catenin, that disrupts its binding to α-catenin, and application of an inhibitor for actin regulator non-muscle Myosin II, phenocopied the effects of the CB1R agonist and inverse agonist on growth cone filopodia, respectively. These findings suggest that both destablization and stabilization of growth cone filopodia are required for RGC axonal fasciculation/defasciculation in the optic tract and that CB1R regulates growth cone filopodia and axon dispersion of RGCs by oppositely modulating β-catenin adhesive and Myosin II actin regulatory functions. This study extends and confirms our understanding of cannabinoid mechanisms in sculpting developing neuronal circuits in vivo., (© 2022 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Limb regeneration is a frontier in biomedical science. Identifying triggers of innate morphogenetic responses in vivo to induce the growth of healthy patterned tissue would address the needs of millions of patients, from diabetics to victims of trauma. Organisms such as Xenopus laevis -whose limited regenerative capacities in adulthood mirror those of humans-are important models with which to test interventions that can restore form and function. Here, we demonstrate long-term (18 months) regrowth, marked tissue repatterning, and functional restoration of an amputated X. laevis hindlimb following a 24-hour exposure to a multidrug, pro-regenerative treatment delivered by a wearable bioreactor. Regenerated tissues composed of skin, bone, vasculature, and nerves significantly exceeded the complexity and sensorimotor capacities of untreated and control animals' hypomorphic spikes. RNA sequencing of early tissue buds revealed activation of developmental pathways such as Wnt/β-catenin, TGF-β, hedgehog, and Notch. These data demonstrate the successful "kickstarting" of endogenous regenerative pathways in a vertebrate model.

In early embryogenesis of fast cleaving embryos, DNA synthesis is short and surveillance mechanisms preserving genome integrity are inefficient, implying the possible generation of mutations. We have analyzed mutagenesis in Xenopus laevis and Drosophila melanogaster early embryos. We report the occurrence of a high mutation rate in Xenopus and show that it is dependent upon the translesion DNA synthesis (TLS) master regulator Rad18. Unexpectedly, we observed a homology-directed repair contribution of Rad18 in reducing the mutation load. Genetic invalidation of TLS in the pre-blastoderm Drosophila embryo resulted in reduction of both the hatching rate and single-nucleotide variations on pericentromeric heterochromatin in adult flies. Altogether, these findings indicate that during very early Xenopus and Drosophila embryos TLS strongly contributes to the high mutation rate. This may constitute a previously unforeseen source of genetic diversity contributing to the polymorphisms of each individual with implications for genome evolution and species adaptation., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Heterologous expression of connexins and innexins in Xenopus oocytes is a powerful approach for studying the biophysical properties of gap junctions (GJs). However, this approach is technically challenging because it requires a differential voltage clamp of two opposed oocytes sharing a common ground. Although a small number of labs have succeeded in performing this technique, essentially all of them have used either homemade amplifiers or commercial amplifiers that were designed for single-oocyte recordings. It is often challenging for other labs to implement this technique. Although a high side current measuring mode has been incorporated into a commercial amplifier for dual oocyte voltage-clamp recordings, there had been no report for its application until our recent study. We have made the high side current measuring approach more practical and convenient by introducing several technical modifications, including the construction of a magnetically based recording platform that allows precise placement of oocytes and various electrodes, use of the bath solution as a conductor in voltage differential electrodes, adoption of a commercial low-leakage KCl electrode as the reference electrode, fabrication of current and voltage electrodes from thin-wall glass capillaries, and positioning of all the electrodes using magnetically based devices. The method described here allows convenient and robust recordings of junctional current (Ij) between two opposed Xenopus oocytes.

Retinoic acid (RA) is a major regulatory signal during embryogenesis produced from vitamin A (retinol) by an extensive, autoregulating metabolic and signaling network to prevent fluctuations that result in developmental malformations. Xenopus laevis is an allotetraploid hybrid frog species whose genome includes L (long) and S (short) chromosomes from the originating species. Evolutionarily, the X. laevis subgenomes have been losing either L or S homoeologs in about 43% of genes to generate singletons. In the RA network, out of the 47 genes, about 47% have lost one of the homoeologs, like the genome average. Interestingly, RA metabolism genes from storage (retinyl esters) to retinaldehyde production exhibit enhanced gene loss with 75% singletons out of 28 genes. The effect of this gene loss on RA signaling autoregulation was studied. Employing transient RA manipulations, homoeolog gene pairs were identified in which one homoeolog exhibits enhanced responses or looser regulation than the other, while in other pairs both homoeologs exhibit similar RA responses. CRISPR/Cas9 targeting of individual homoeologs to reduce their activity supports the hypothesis where the RA metabolic network gene loss results in tighter network regulation and more efficient RA robustness responses to overcome complex regulation conditions.

Vertebrates evolved mechanisms for sodium conservation and gas exchange in conjunction with migration from aquatic to terrestrial habitats. Epithelial Na + channel (ENaC) function is critical to systems responsible for extracellular fluid homeostasis and gas exchange. ENaC is activated by cleavage at multiple specific extracellular polybasic sites, releasing inhibitory tracts from the channel's α and γ subunits. We found that proximal and distal polybasic tracts in ENaC subunits coevolved, consistent with the dual cleavage requirement for activation observed in mammals. Polybasic tract pairs evolved with the terrestrial migration and the appearance of lungs, coincident with the ENaC activator aldosterone, and appeared independently in the α and γ subunits. In summary, sites within ENaC for protease activation developed in vertebrates when renal Na + conservation and alveolar gas exchange were required for terrestrial survival., Competing Interests: XW, DB, CG, NC, OK No competing interests declared, (© 2022, Wang et al.)

Amphibian metamorphosis is a transitional period that involves significant changes in the cell-type populations and biological processes occurring in the brain. Analysis of gene expression dynamics during this process may provide insight into the molecular events underlying these changes. We conducted differential gene expression analyses of the developing Xenopus laevis tadpole brain during this period in two ways: first, over stages of the development in the midbrain and, second, across regions of the brain at a single developmental stage. We found that genes pertaining to positive regulation of neural progenitor cell proliferation as well as known progenitor cell markers were upregulated in the midbrain prior to metamorphic climax; concurrently, expression of cell cycle timing regulators decreased across this period, supporting the notion that cell cycle lengthening contributes to a decrease in proliferation by the end of metamorphosis. We also found that at the start of metamorphosis, neural progenitor populations appeared to be similar across the fore-, mid-, and hindbrain regions. Genes pertaining to negative regulation of differentiation were upregulated in the spinal cord compared to the rest of the brain, however, suggesting that different programs may regulate neurogenesis there. Finally, we found that regulation of biological processes like cell fate commitment and synaptic signaling follow similar trajectories in the brain across early tadpole metamorphosis and mid- to late-embryonic mouse development. By comparing expression across both temporal and spatial conditions, we have been able to illuminate cell-type and biological pathway dynamics in the brain during metamorphosis., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

Apical constriction refers to the active, actomyosin-driven process that reduces apical cell surface area in epithelial cells. Apical constriction is utilized in epithelial morphogenesis during embryonic development in multiple contexts, such as gastrulation, neural tube closure, and organogenesis. Defects in apical constriction can result in congenital birth defects, yet our understanding of the molecular control of apical constriction is relatively limited. To uncover new genetic regulators of apical constriction and gain mechanistic insight into the cell biology of this process, we need reliable assay systems that allow real-time observation and quantification of apical constriction as it occurs and permit gain- and loss-of-function analyses to explore gene function and interaction during apical constriction. In this chapter, we describe using the early Xenopus embryo as an assay system to investigate molecular mechanisms involved in apical constriction during both gastrulation and neurulation., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

In neurons, specific mRNAs are transported into axons, where their local translation supports essential cellular functions. Over the years, our knowledge of the molecular mechanisms underlying axonal mRNA translation has rapidly expanded. However, tools to study mRNA localization and translation in real time with high spatial precision were not available until recently. Here, we present a live imaging approach to examine axonal mRNA trafficking and translation simultaneously in Xenopus retinal ganglion cells (RGCs), using in vitro synthesized fluorescently labeled mRNAs coupled with a genetically encoded protein tagging system to visualize synthesizing peptides at single-molecule resolution. We further describe the process of image analysis in detail, thus providing a methodology that can be used to investigate new research questions in the field., (© 2022. The Author(s).)

Planar cell polarity (PCP) refers to coordinated cell polarization in the plane of the tissue. Genetic studies in Drosophila identified several core PCP genes, whose products function together in a signaling pathway that regulates cell shape, epithelial tissue organization and remodeling during morphogenesis. PCP is detected by the asymmetric distribution of core PCP proteins at different borders of epithelial cells. Believed to be critical for signaling, this segregation is studied by a variety of techniques, such as direct immunostaining and imaging of fluorescent PCP protein fusions or fluorescence recovery after photobleaching (FRAP). All of the above techniques can be applied to the analysis of the Xenopus neural plate to study the dynamics of tissue polarization, making this system one of the best vertebrate PCP models. This chapter describes how to image PCP proteins in Xenopus neuroectoderm for both fixed and live samples. These robust cellular techniques will contribute to mechanistic studies of PCP in vertebrate embryos., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Boric acid is a vital micronutrient that is toxic at high concentrations in animals. However, the mechanisms underlying boric acid transport in animal cells remain unclear. To identify the plasma membrane boric acid channels in animals, we analyzed the function of human aquaporins (AQPs), which are homologous to the nodulin-like intrinsic protein family of plant boric acid channels. When human AQPs were expressed in Xenopus laevis oocytes, the results of the swelling assay showed that boric acid permeability significantly increased in oocytes expressing AQP3, 7, 8, 9, and 10, but not in those expressing AQP1, 2, 4, and 5. The boric acid influxes of these oocytes were also confirmed by elemental quantification. Electrophysiological analysis using a pH microelectrode showed that these AQPs transported boric acid (B(OH) 3 ) but not borate ions (B(OH) 4 - ). These results indicate that AQP3, 7, 8, 9, and 10 act as boric acid transport systems, likely as channels in humans., (© 2022 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

The nuclear pore complex (NPC) is the conduit in the nuclear envelope through which proteins and RNA are transported between the cytoplasm and nucleus. Xenopus egg extracts that support de novo assembly of nuclei have provided a robust system to study NPC structure and function because the biochemical composition of the extract can be easily manipulated. Here we describe how to assemble nuclei in Xenopus egg extract, how to visualize and analyze NPCs in both live and fixed samples, and different approaches to altering nucleocytoplasmic transport in extract., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Thyroid hormone (T3) affects many diverse physiological processes such as metabolism, organogenesis, and growth. The two highly related frog species, diploid Xenopus tropicalis and pseudo tetraploid Xenopus laevis, have been used as models for analyzing the effects of T3 during vertebrate development. T3 regulates T3-inducible gene transcription through T3 receptor (TR)-binding to T3-response elements (TREs). We have previously identified sperm associated antigen 7 (spag7) as a candidate T3 target gene that is potentially involved in adult stem cell development and/or proliferation during intestinal metamorphosis. To investigate whether T3 regulates spag7 directly at the transcriptional level via TR, we first conducted qRT-PCR to analyze its expression during natural and T3-induced metamorphosis and found that spag7 was up-regulated during natural metamorphosis in the intestine, tail, brain and hindlimb, peaking at the climax of metamorphosis in all those organs, and upon T3 treatment of premetamorphic tadpoles. Next, we demonstrated that an intronic TRE in spag7, first identified through bioinformatic analysis, could bind to TR in vitro and in vivo during metamorphosis. A dual luciferase assay utilizing a reconstituted frog oocyte transcription system showed that the TRE could mediate promoter activation by liganded TR. These results indicate that spag7 expression is directly regulated by T3 through the TRE in the first intron during metamorphosis, implicating a role for spag7 early during T3-regulated tissue remodeling and resorption., (© Published 2021. This article is a U.S. Government work and is in the public domain in the USA.)

P2X7 receptors (P2X7Rs) are fast ATP 4- -gated ion channels that, like other members of the P2X receptor family, function as homotrimers. A high-resolution cryo-EM structure of the full-length rat P2X7R is available. Using voltage-clamp experiments in Xenopus laevis oocytes, even the earliest steps of P2X7R activation can be quantitatively recorded in the millisecond range. Site-directed mutagenesis combined with voltage-clamp recordings can reveal residues and domains of the P2X7R involved in ATP 4- binding, gating (i.e., opening and closing of the channel pore) and ion selectivity. We present here proven voltage-clamp protocols that take into account requirements that are important at the levels of cDNA and vector sequences, cRNA synthesis, and Xenopus laevis oocyte isolation for reliable results., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The long intracellular P2X7 C-terminus accounts for diverse downstream effects of P2X7 activation. Although the recent determination of the cryo-EM structure of the full-length P2X7 receptor finally revealed the structure and several unexpected features of the large cytoplasmic domain, its molecular function remains enigmatic. Incorporation of unnatural amino acids (UAA) via an amber Stop codon has been a powerful tool for structure-function analysis of proteins. Voltage clamp fluorometry (VCF) with the fluorescent unnatural amino acid L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (ANAP) provides a means to study intracellular domain movements of ion channel receptors. In the Xenopus laevis oocyte expression system, site-specific introduction of this environment-sensitive fluorophore can be achieved by the nuclear injection of cDNA encoding an orthogonal amber suppressor tRNA/aminoacyl-tRNA synthetase pair and subsequent cytoplasmic injection of ANAP together with the respective cRNA containing the amber Stop codon. Here, we describe this protocol for expression of ANAP-labeled P2X7. In addition, we provide a simplified alternative protocol, in which we coinject cRNAs encoding the tRNA synthetase and mutant P2X7 together with the synthesized amber suppressor tRNA and ANAP in one step into the cytosol. We found that the new protocol yielded more reproducible results and was less harmful for the oocytes. By selective fluorescence labeling of the ANAP-labeled P2X7 protein in the oocyte plasma membrane and VCF recordings, we show that this method results in comparable levels of functional ANAP-labeled P2X7 protein., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In this study we explore the sub-lethal effects of two malaria vector control pesticides, deltamethrin and dichlorodiphenyltrichloroethane (DDT), on Xenopus laevis by incorporating different levels of biological organisation. Pesticide accumulation in frog tissue was measured alongside liver metabolomics and individual swimming behaviour to assess whether changes presented at these different levels, and if such changes could be linked between levels. Results showed evidence of concentration dependent accumulation of DDT and its metabolites, but no measurable accumulation of deltamethrin in adult X. laevis after 96 h of exposure. Both DDT and deltamethrin were shown to cause alterations in the liver metabolome of X. laevis. We also showed that some of these changes can be enhanced in exposure to a mixture of these two pesticides. Initial behavioural responses recorded directly after exposure were seen in the form of decreased activity, less alterations between mobility states, and less time spent at the water surface. This response persisted after 96 h of exposure to a mixture of the two pesticides. This study shows that sub-lethal exposure to pesticides can alter the biochemical homeostasis of frogs with the potential to cascade onto behavioural and ecological levels in mixture exposure scenarios., (Copyright © 2021. Published by Elsevier Inc.)

Cajal-Retzius cells are essential for cortical development in mammals, and their involvement in the evolution of this structure has been widely postulated, but very little is known about their progenitor domains in non-mammalian vertebrates. Using in situhybridization and immunofluorescence techniques we analyzed the expression of some of the main Cajal-Retzius cell markers such as Dbx1, Ebf3, ER81, Lhx1, Lhx5, p73, Reelin, Wnt3a, Zic1, and Zic2 in the forebrain of the anuran Xenopus laevis, because amphibians are the only class of anamniote tetrapods and show a tetrapartite evaginated pallium, but no layered or nuclear organization. Our results suggested that the Cajal-Retzius cell progenitor domains were comparable to those previously described in amniotes. Thus, at dorsomedial telencephalic portions a region comparable to the cortical hem was defined in Xenopus based on the expression of Wnt3a, p73, Reelin, Zic1, and Zic2. In the septum, two different domains were observed: a periventricular dorsal septum, at the limit between the pallium and the subpallium, expressing Reelin, Zic1, and Zic2, and a related septal domain, expressing Ebf3, Zic1, and Zic2. In the lateral telencephalon, the ventral pallium next to the pallio-subpallial boundary, the lack of Dbx1 and the unique expression of Reelin during development defined this territory as the most divergent with respect to mammals. Finally, we also analyzed the expression of these markers at the prethalamic eminence region, suggested as Cajal-Retzius progenitor domain in amniotes, observing there Zic1, Zic2, ER81, and Lhx1 expression. Our data show that in anurans there are different subtypes and progenitor domains of Cajal-Retzius cells, which probably contribute to the cortical regional specification and territory-specific properties. This supports the notion that the basic organization of pallial derivatives in vertebrates follows a comparable fundamental arrangement, even in those that do not have a sophisticated stratified cortical structure like the mammalian cerebral cortex., (© 2021 S. Karger AG, Basel.)

Benzodiazepines (BZDs) produce versatile pharmacological actions through positive modulation of GABA A receptors (GABA A Rs). A previous study has demonstrated that high concentrations of diazepam potentiate GABA currents on the α 1 β 2 γ 2 and α 1 β 2 GABA A Rs in a flumazenil-insensitive manner. In this study, the high-concentration effects of BZDs and their sensitivity to flumazenil were determined on synaptic (α 1 β 2 γ 2 , α 2 β 2 γ 2 , α 5 β 2 γ 2 ) and extra-synaptic (α 4 β 2 δ) GABA A Rs using the voltage-clamp electrophysiology technique. The in vivo evaluation of flumazenil-insensitive BZD effects was conducted in mice via the loss of righting reflex (LORR) test. Diazepam induced biphasic potentiation on the α 1 β 2 γ 2 , α 2 β 2 γ 2 and α 5 β 2 γ 2 GABA A Rs, but did not affect the α 4 β 2 δ receptor. In contrast to the nanomolar component of potentiation, the second potentiation elicited by micromolar diazepam was insensitive to flumazenil. Midazolam, clonazepam, and lorazepam at 200 µM exhibited similar flumazenil-insensitive effects on the α 1 β 2 γ 2 , α 2 β 2 γ 2 and α 5 β 2 γ 2 receptors, whereas the potentiation induced by 200 µM zolpidem or triazolam was abolished by flumazenil. Both the GABA A R antagonist pentylenetetrazol and Fa173, a proposed transmembrane site antagonist, abolished the potentiation induced by 200 µM diazepam. Consistent with the in vitro results, flumazenil antagonized the zolpidem-induced LORR, but not that induced by diazepam or midazolam. Pentylenetetrazol and Fa173 antagonized the diazepam-induced LORR. These findings support the existence of non-classical BZD binding sites on certain GABA A R subtypes and indicate that the flumazenil-insensitive effects depend on the chemical structures of BZD ligands.

When vertebrate replisomes from neighboring origins converge, the Mcm7 subunit of the replicative helicase, CMG, is ubiquitylated by the E3 ubiquitin ligase, CRL2Lrr1. Polyubiquitylated CMG is then disassembled by the p97 ATPase, leading to replication termination. To avoid premature replisome disassembly, CRL2Lrr1 is only recruited to CMGs after they converge, but the underlying mechanism is unclear. Here, we use cryogenic electron microscopy to determine structures of recombinant Xenopus laevis CRL2Lrr1 with and without neddylation. The structures reveal that CRL2Lrr1 adopts an unusually open architecture, in which the putative substrate-recognition subunit, Lrr1, is located far from the catalytic module that catalyzes ubiquitin transfer. We further demonstrate that a predicted, flexible pleckstrin homology domain at the N-terminus of Lrr1 is essential to target CRL2Lrr1 to terminated CMGs. We propose a hypothetical model that explains how CRL2Lrr1's catalytic module is positioned next to the ubiquitylation site on Mcm7, and why CRL2Lrr1 binds CMG only after replisomes converge., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Neonicotinoids (NEO) represent the main class of insecticides currently in use, with thiamethoxam (THX) and clothianidin (CLO) primarily applied agriculturally. With few comprehensive studies having been performed with non-target amphibians, the aim was to investigate potential biomarker responses along an adverse outcome pathway of NEO exposure, whereby data were collected on multiple biological hierarchies. Juvenile African clawed frogs, Xenopus laevis , were exposed to commercial formulations of THX and CLO at high (100 ppm) and low (20 ppm) concentrations of the active ingredient. Mortality, growth, development, liver metabolic enzyme activity, and gene expression endpoints were quantified. Tadpoles ( n > 1000) from NF 47 through tail resorption stage (NF 66) were exposed to NEO or to NEO-free media treatments. Liver cell reductase activity and cytotoxicity were quantified by flow cytometry. Compared to control reference gene expressions, levels of expression for NEO receptor subunits, cell structure, function, and decontamination processes were measured by RT-qPCR by using liver and brain. Mortality in THX high was 21.5% compared to the control (9.1%); the metabolic conversion of THX to CLO may explain these results. The NF 57 control tadpoles were heavier, longer, and more developed than the others. The progression of development from NF 57-66 was reduced by THX low, and weight gain was impaired. Liver reductases were highest in the control (84.1%), with low NEO exhibiting the greatest reductions; the greatest cytotoxicity was seen with THX high. More transcriptional activity was noted in brains than in livers. Results affirm the utility of a study approach that considers multiple complexities in ecotoxicological studies with non-target amphibians, underscoring the need for simultaneously considering NEO concentration-response relationships with both whole-organism and biomarker endpoints.

All living systems perpetuate themselves via growth in or on the body, followed by splitting, budding, or birth. We find that synthetic multicellular assemblies can also replicate kinematically by moving and compressing dissociated cells in their environment into functional self-copies. This form of perpetuation, previously unseen in any organism, arises spontaneously over days rather than evolving over millennia. We also show how artificial intelligence methods can design assemblies that postpone loss of replicative ability and perform useful work as a side effect of replication. This suggests other unique and useful phenotypes can be rapidly reached from wild-type organisms without selection or genetic engineering, thereby broadening our understanding of the conditions under which replication arises, phenotypic plasticity, and how useful replicative machines may be realized., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

The Spemann and Mangold Organizer (SMO) is of fundamental importance for dorsal ventral body axis formation during vertebrate embryogenesis. Maternal Huluwa (Hwa) has been identified as the dorsal determinant that is both necessary and sufficient for SMO formation. However, it remains unclear how Hwa is regulated. Here, we report that the E3 ubiquitin ligase zinc and ring finger 3 (ZNRF3) is essential for restricting the spatial activity of Hwa and therefore correct SMO formation in Xenopus laevis. ZNRF3 interacts with and ubiquitinates Hwa, thereby regulating its lysosomal trafficking and protein stability. Perturbation of ZNRF3 leads to the accumulation of Hwa and induction of an ectopic axis in embryos. Ectopic expression of ZNRF3 promotes Hwa degradation and dampens the axis-inducing activity of Hwa. Thus, our findings identify a substrate of ZNRF3, but also highlight the importance of the regulation of Hwa temporospatial activity in body axis formation in vertebrate embryos., (© 2021 The Authors.)

Mutations of TRPV3 lead to severe dermal hyperkeratosis in Olmsted syndrome, but whether the mutants are trafficked to the cell membrane or not is controversial. Even less is known about TRPV3 function in intestinal epithelia, although research on ruminants and pigs suggests an involvement in the uptake of NH 4 + . It was the purpose of this study to measure the permeability of the human homologue (hTRPV3) to NH 4 + , to localize hTRPV3 in human skin equivalents, and to investigate trafficking of the Olmsted mutant G573S. Immunoblotting and immunostaining verified the successful expression of hTRPV3 in HEK-293 cells and Xenopus oocytes with trafficking to the cell membrane. Human skin equivalents showed distinct staining of the apical membrane of the top layer of keratinocytes with cytosolic staining in the middle layers. Experiments with pH-sensitive microelectrodes on Xenopus oocytes demonstrated that acidification by NH 4 + was significantly greater when hTRPV3 was expressed. Single-channel measurements showed larger conductances in overexpressing Xenopus oocytes than in controls. In whole-cell experiments on HEK-293 cells, both enantiomers of menthol stimulated influx of NH 4 + in hTRPV3 expressing cells, but not in controls. Expression of the mutant G573S greatly reduced cell viability with partial rescue via ruthenium red. Immunofluorescence confirmed cytosolic expression, with membrane staining observed in a very small number of cells. We suggest that expression of TRPV3 by epithelia may have implications not just for Ca 2+ signalling, but also for nitrogen metabolism. Models suggesting how influx of NH 4 + via TRPV3 might stimulate skin cornification or intestinal NH 4 + transport are discussed., (© 2021. The Author(s).)

The importance of histone variant H2A.Z in transcription regulation has been well established, yet its mechanism-of-action remains enigmatic. Conflicting evidence exists in support of both an activating and a repressive role of H2A.Z in transcription. Here we report cryo-electron microscopy (cryo-EM) structures of nucleosomes and chromatin fibers containing H2A.Z and those containing canonical H2A. The structures show that H2A.Z incorporation results in substantial structural changes in both nucleosome and chromatin fiber. While H2A.Z increases the mobility of DNA terminus in nucleosomes, it simultaneously enables nucleosome arrays to form a more regular and condensed chromatin fiber. We also demonstrated that H2A.Z's ability to enhance nucleosomal DNA mobility is largely attributed to its characteristic shorter C-terminus. Our study provides the structural basis for H2A.Z-mediated chromatin regulation, showing that the increase flexibility of the DNA termini in H2A.Z nucleosomes is central to its dual-functions in chromatin regulation and in transcription., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Photolyases (PHRs) repair ultraviolet (UV)-induced DNA photoproducts into normal bases. In this study, we measured the conformational changes upon photoactivation and photorepair processes of a PHR and its specific substrates, (6-4)PHR and a pyrimidine(6-4)pyrimidone photoproduct ((6-4)PP), by light-induced difference Fourier transform infrared (FT-IR) spectroscopy. The single-stranded DNA with (6-4)PP (ss(6-4)PP) was used as a substrate and the resultant FT-IR spectra were compared with the previous results on double-stranded DNA with (6-4)PP (ds(6-4)PP). In the excess amount of substrate to the enzyme, different ss(6-4)PP photorepair FT-IR signals were obtained in an illumination time-dependent manner. As reported for ds(6-4)PP, the early stages of the photoreaction involve the changes in the ss(6-4)PP only, while the late stages of the reaction involve the ss(6-4)PP repair-associated changes and dissociation from (6-4)PHR. From these spectra, difference spectra originating from the binding/dissociation spectrum were extracted. The signals of the C═O stretches of (6-4)PP and repaired thymines in the single- and double-stranded DNA were tentatively assigned. The C═O stretches of (6-4)PP were observed at frequencies that reflect single- and double-stranded DNA environments in aqueous solution, reflecting the different hydrogen-bonding environments. The conformational changes of PHR upon binding of ss(6-4)PP and ds(6-4)PP were similar, suggesting that the conformational change is limited to the (6-4)PP binding pocket region. We interpreted that ds(6-4)PP may be bound together without any special mechanism for flipping out.

The high-affinity tyrosine kinase receptor MET plays a pivotal role in several facets of cell regulation. Although its mitogenic effect is well documented, some aspects of connection patterns between signaling pathways involved in cell cycle progression remain to be deciphered. We have used a tractable heterologous expression system, the Xenopus oocyte, to detect connections between distinct MET signaling cascades involved in G2/M progression. Our results reveal that Src acts as an adapter via its SH2 domain to recruit 3-phosphoinositide-dependent protein kinase 1 (PDK1) to the MET signaling complex leading to Akt phosphorylation. These data define an original crosstalk between Src and Akt signaling pathways that contributes to MET-induced entry into the M phase, and deserves further investigation in pathologies harboring deregulation of this receptor., (© 2021 Federation of European Biochemical Societies.)

Meiosis facilitates diversity across individuals and serves as a major driver of evolution. However, understanding how meiosis begins is complicated by fundamental differences that exist between sexes and species. Fundamental meiotic research is further hampered by a current lack of human meiotic cells lines. Consequently, much of what we know relies on data from model organisms. However, contextualising findings from yeast, worms, flies and mice can be challenging, due to marked differences in both nomenclature and the relative timing of meiosis. In this review, we set out to combine current knowledge of signalling and transcriptional pathways that control meiosis initiation across the sexes in a variety of organisms. Furthermore, we highlight the emerging links between meiosis initiation and oncogenesis, which might explain the frequent re-expression of normally silent meiotic genes in a variety of human cancers., (© 2021 The Author(s).)

The gene regulatory networks that coordinate the development of the cardiac and pulmonary systems are essential for terrestrial life but poorly understood. The T-box transcription factor Tbx5 is critical for both pulmonary specification and heart development, but how these activities are mechanistically integrated remains unclear. Here using Xenopus and mouse embryos, we establish molecular links between Tbx5 and retinoic acid (RA) signaling in the mesoderm and between RA signaling and sonic hedgehog expression in the endoderm to unveil a conserved RA-Hedgehog-Wnt signaling cascade coordinating cardiopulmonary (CP) development. We demonstrate that Tbx5 directly maintains expression of aldh1a2, the RA-synthesizing enzyme, in the foregut lateral plate mesoderm via an evolutionarily conserved intronic enhancer. Tbx5 promotes posterior second heart field identity in a positive feedback loop with RA, antagonizing a Fgf8-Cyp regulatory module to restrict FGF activity to the anterior. We find that Tbx5/Aldh1a2-dependent RA signaling directly activates shh transcription in the adjacent foregut endoderm through a conserved MACS1 enhancer. Hedgehog signaling coordinates with Tbx5 in the mesoderm to activate expression of wnt2/2b, which induces pulmonary fate in the foregut endoderm. These results provide mechanistic insight into the interrelationship between heart and lung development informing CP evolution and birth defects., Competing Interests: SR, JS, XY, AR, KA, PC, KI, MH, IM, AZ No competing interests declared, (© 2021, Rankin et al.)

The enpp ectonucleotidases regulate lipidic and purinergic signalling pathways by controlling the extracellular concentrations of purines and bioactive lipids. Although both pathways are key regulators of kidney physiology and linked to human renal pathologies, their roles during nephrogenesis remain poorly understood. We previously showed that the pronephros was a major site of enpp expression and now demonstrate an unsuspected role for the conserved vertebrate enpp4 protein during kidney formation in Xenopus. Enpp4 over-expression results in ectopic renal tissues and, on rare occasion, complete mini-duplication of the entire kidney. Enpp4 is required and sufficient for pronephric markers expression and regulates the expression of RA, Notch and Wnt pathway members. Enpp4 is a membrane protein that binds, without hydrolyzing, phosphatidylserine and its effects are mediated by the receptor s1pr5, although not via the generation of S1P. Finally, we propose a novel and non-catalytic mechanism by which lipidic signalling regulates nephrogenesis., (© 2021. The Author(s).)

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.)