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Recent research has shown that membrane trafficking plays an important role in canonical Wnt signaling through sequestration of the β-catenin destruction complex inside multivesicular bodies (MVBs) and lysosomes. In this study, we introduce Ouabain, an inhibitor of the Na,K-ATPase pump that establishes electric potentials across membranes, as a potent inhibitor of Wnt signaling. We find that Na,K-ATPase levels are elevated in advanced colon carcinoma, that this enzyme is elevated in cancer cells with constitutively activated Wnt pathway and is activated by GSK3 inhibitors that increase macropinocytosis. Ouabain blocks macropinocytosis, which is an essential step in Wnt signaling, probably explaining the strong effects of Ouabain on this pathway. In Xenopus embryos, brief Ouabain treatment at the 32-cell stage, critical for the earliest Wnt signal in development-inhibited brains, could be reversed by treatment with Lithium chloride, a Wnt mimic. Inhibiting membrane trafficking may provide a way of targeting Wnt-driven cancers.

Neural crest cells exemplify cellular diversification from a multipotent progenitor population. However, the full sequence of early molecular choices orchestrating the emergence of neural crest heterogeneity from the embryonic ectoderm remains elusive. Gene-regulatory-networks (GRN) govern early development and cell specification toward definitive neural crest. Here, we combine ultradense single-cell transcriptomes with machine-learning and large-scale transcriptomic and epigenomic experimental validation of selected trajectories, to provide the general principles and highlight specific features of the GRN underlying neural crest fate diversification from induction to early migration stages using Xenopus frog embryos as a model. During gastrulation, a transient neural border zone state precedes the choice between neural crest and placodes which includes multiple converging gene programs. During neurulation, transcription factor connectome, and bifurcation analyses demonstrate the early emergence of neural crest fates at the neural plate stage, alongside an unbiased multipotent-like lineage persisting until epithelial-mesenchymal transition stage. We also decipher circuits driving cranial and vagal neural crest formation and provide a broadly applicable high-throughput validation strategy for investigating single-cell transcriptomes in vertebrate GRNs in development, evolution, and disease.

Frogs are an ecologically diverse and phylogenetically ancient group of anuran amphibians that include important vertebrate cell and developmental model systems, notably the genus Xenopus. Here we report a high-quality reference genome sequence for the western clawed frog, Xenopus tropicalis, along with draft chromosome-scale sequences of three distantly related emerging model frog species, Eleutherodactylus coqui, Engystomops pustulosus, and Hymenochirus boettgeri. Frog chromosomes have remained remarkably stable since the Mesozoic Era, with limited Robertsonian (i.e., arm-preserving) translocations and end-to-end fusions found among the smaller chromosomes. Conservation of synteny includes conservation of centromere locations, marked by centromeric tandem repeats associated with Cenp-a binding surrounded by pericentromeric LINE/L1 elements. This work explores the structure of chromosomes across frogs, using a dense meiotic linkage map for X. tropicalis and chromatin conformation capture (Hi-C) data for all species. Abundant satellite repeats occupy the unusually long (~20 megabase) terminal regions of each chromosome that coincide with high rates of recombination. Both embryonic and differentiated cells show reproducible associations of centromeric chromatin and of telomeres, reflecting a Rabl-like configuration. Our comparative analyses reveal 13 conserved ancestral anuran chromosomes from which contemporary frog genomes were constructed.

Background: Regenerative medicine and transplantation science continuously seek methods to circumvent immune-mediated rejection and promote tissue regeneration. Sertoli cells, with their inherent immunoprotective properties, emerge as pivotal players in this quest. However, whether Sertoli cells can play immunomodulatory role in tadpole tail regeneration and can thus benefit the regeneration process are needed to be discovered. Methods: Immature Sertoli cells from Xenopus tropicalis (XtiSCs) were transplanted into X. tropicalis tadpoles, followed by the amputation of the final third of their tails. We assessed the migration of XtiSCs, tail regeneration length, muscle degradation and growth, and macrophage counts across various regions including the entire tail, tail trunk, injection site, and regeneration site. The interactions between XtiSCs and macrophages were examined using a confocal microscope. To deplete macrophages, clodronate liposomes were administered prior to the transplantation of XtiSCs, while the administration of control liposomes acted as a negative control. Student's t-test was used to compare the effects of XtiSCs injection to those of a 2/3PBS injection across groups with no liposomes, control liposomes, and clodronate liposomes. Results: XtiSCs have excellent viability after transplantation to tadpole tail and remarkable homing capabilities to the regeneration site after tail amputation. XtiSCs injection increased macrophage numbers at 3 days post-amputation and 5 days post-amputation in the tail trunk, specifically at the injection site and at the regenerated tail, in a macrophage depleted environment (clodronate-liposome injection). What's more, XtiSCs injection decreased muscle fibers degradation significantly at 1 day post-amputation and facilitated new muscle growth significantly at 3 days post-amputation. In addition, whole-mount immunostaining showed that some XtiSCs co-localized with macrophages. And we observed potential mitochondria transport from XtiSCs to macrophages using MitoTracker staining in tadpole tail. Conclusions: Our study delineates the novel role of XtiSCs in facilitating muscle regeneration post tadpole tail amputation, underscoring a unique interaction with macrophages that is crucial for regenerative success. This study not only highlights the therapeutic potential of Sertoli cells in regenerative medicine but also opens avenues for clinical translation, offering insights into immunoregulatory strategies that could enhance tissue regeneration and transplant acceptance. [ABSTRACT FROM AUTHOR]

This review explores the regenerative capacity of Xenopus laevis, focusing on tail regeneration, as a model to uncover cellular, molecular, and developmental mechanisms underlying tissue repair. X. laevis tadpoles provide unique insights into regenerative biology due to their regeneration-competent and -incompetent stages and ability to regrow complex structures in the tail, including the spinal cord, muscle, and skin, after amputation. The review delves into the roles of key signaling pathways, such as those involving reactive oxygen species (ROS) and signaling molecules like BMPs and FGFs, in orchestrating cellular responses during regeneration. It also examines how mechanotransduction, epigenetic regulation, and metabolic shifts influence tissue restoration. Comparisons of regenerative capacity with other species shed light on the evolutionary loss of regenerative abilities and underscore X. laevis as an invaluable model for understanding the constraints of tissue repair in higher organisms. This comprehensive review synthesizes recent findings, suggesting future directions for exploring regeneration mechanisms, with potential implications for advancing regenerative medicine. [ABSTRACT FROM AUTHOR]

The olfactory organ of tetrapods, with few exceptions, comprises the main and accessory organs: olfactory epithelium (OE) and vomeronasal organ (VNO). Unlike tetrapods, teleost fish lack a VNO. However, lungfish, a type of sarcopterygian fish closely related to tetrapods, possesses a lamellar OE similar to the OE of teleosts and a recess epithelium (RecE) resembling the amphibian VNO. The RecE has been hypothesized as a primordial VNO. Olfactory receptors in tetrapods are distinctively expressed in the OE and VNO. For instance, type 2 vomeronasal receptors (V2Rs) in Xenopus are categorized into those exclusively expressed in the OE and those solely expressed in the VNO. It remains unclear whether V2Rs are differentially expressed between the lamellar OE and RecE in lungfish. This study investigated V2R expression in the lamellar OE and RecE of the African lungfish, Protopterus annectens. P. annectens V2Rs were categorized into three groups: those exclusively expressed in the lamellar OE, those exclusively expressed in the RecE, and those expressed in both the lamellar OE and RecE. V2Rs exclusively expressed in the RecE and those expressed in both the lamellar OE and RecE formed a distinct clade in the phylogenetic tree, whereas others were solely expressed in the lamellar OE. These findings suggest that lungfish V2R expression represents an intermediate stage toward complete segregation between V2Rs expressed in the OE and those expressed in the VNO. [ABSTRACT FROM AUTHOR]

Hepatic enzyme induction, an inherent defense system against xenobiotics, is known to simultaneously affect endocrine system functions in mammals under specific conditions, particularly thyroid hormone (TH) regulation. While this phenomenon has been studied extensively, the pathway leading to this indirect thyroid effect in mammals has unclear applicability to amphibians, despite the importance of amphibian species in assessing thyroid‐disruptive chemicals. Here, we investigated the effects of three well‐known mammalian enzyme inducers—β‐naphthoflavone (BNF), pregnenolone carbonitrile (PCN), and sodium phenobarbital (NaPB)—on the gene expression of phase‐I and phase‐II metabolizing enzymes in Xenopus laevis tadpoles. Waterborne exposure to BNF and PCN significantly induced the expression of both phase‐I (cytochrome P450, CYP) and phase‐II enzymes (UDP‐glucuronosyltransferase, UGT and sulfotransferase, SULT), but in different patterns, while NaPB exposure induced CYP2B expression without affecting phase‐II enzymes in tadpoles, in contrast to mammals. Furthermore, an ex vivo hepatic enzyme activity assay confirmed that BNF treatment significantly increased phase‐II metabolic activity (glucuronidation and sulfation) toward TH. These results suggest the potential for certain mammalian enzyme inducers to influence TH clearance in X. laevis tadpoles. Our findings provide insights into the profiles of xenosensing activity and enzyme induction in amphibians, which can facilitate a better understanding of the mechanisms of indirect effects on the thyroid system via hepatic enzyme induction in nonmammalian species. This study examined the effects of waterborne exposure to typical mammalian hepatic enzyme inducers—β‐naphthoflavone (BNF), pregnenolone carbonitrile (PCN), and sodium phenobarbital (NaPB)—on Xenopus laevis tadpoles. BNF and PCN significantly upregulated the gene expression of both phase‐I and phase‐II metabolizing enzymes in different patterns, while NaPB only induced CYP2B expression without affecting phase‐II enzymes, unlike in mammals. BNF also enhanced ex vivo phase‐II metabolic activity toward thyroid hormone (TH), suggesting hepatic enzyme induction may influence TH clearance in tadpoles. [ABSTRACT FROM AUTHOR]

The article "GABA transport: beyond stress? A closer look at AtGAT2" published in the Journal of Experimental Botany explores the role of the GABA transporter AtGAT2 in Arabidopsis. GABA is a stress-response compound found in various plant tissues and organs, with AtGAT2 identified as a low-affinity GABA transporter. The study delves into the transport properties of AtGAT2, highlighting its voltage-dependent nature and role in maintaining GABA homeostasis under stress conditions. The research challenges conventional views on GABA transporters and opens avenues for further exploration into the molecular mechanisms underlying GABA transport in plants. [Extracted from the article]

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

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

Maurotoxin (MTX) is a 34-residue peptide from Scorpio maurus venom. It is reticulated by four disulfide bridges with a unique arrangement compared to other scorpion toxins that target potassium (K+) channels. Structure–activity relationship studies have not been well performed for this toxin family. The screening of Scorpio maurus venom was performed by different steps of fractionation, followed by the ELISA test, using MTX antibodies, to isolate an MTX-like peptide. In vitro, in vivo and computational studies were performed to study the structure–activity relationship of the new isolated peptide. We isolated a new peptide designated MTX1, structurally related to MTX. It demonstrated toxicity on mice eight times more effectively than MTX. MTX1 blocks the Kv1.2 and Kv1.3 channels, expressed in Xenopus oocytes, with IC50 values of 0.26 and 180 nM, respectively. Moreover, MTX1 competitively interacts with both 125I-apamin (IC50 = 1.7 nM) and 125I-charybdotoxin (IC50 = 5 nM) for binding to rat brain synaptosomes. Despite its high sequence similarity (85%) to MTX, MTX1 exhibits a higher binding affinity towards the Kv1.2 and SKCa channels. Computational analysis highlights the significance of specific residues in the β-sheet region, particularly the R27, in enhancing the binding affinity of MTX1 towards the Kv1.2 and SKCa channels. [ABSTRACT FROM AUTHOR]

Primordial germ cells (PGCs) are the precursors of sperms and oocytes. Proper development of PGCs is crucial for the survival of the species. In many organisms, factors responsible for PGC development are synthesized during early oogenesis and assembled into the germ plasm. During early embryonic development, germ plasm is inherited by a few cells, leading to the formation of PGCs. While germline development has been extensively studied, how components of the germ plasm regulate PGC development is not fully understood. Here, we report that Dzip1 is dynamically expressed in vertebrate germline and is a novel component of the germ plasm in Xenopus and zebrafish. Knockdown of Dzip1 impairs PGC development in Xenopus embryos. At the molecular level, Dzip1 physically interacts with Dazl, an evolutionarily conserved RNA-binding protein that plays a multifaced role during germline development. We further showed that the sequence between amino acid residues 282 and 550 of Dzip1 is responsible for binding to Dazl. Disruption of the binding between Dzip1 and Dazl leads to defective PGC development. Taken together, our results presented here demonstrate that Dzip1 is dynamically expressed in the vertebrate germline and plays a novel function during Xenopus PGC development. [Display omitted] • Dzip1 is dynamically expressed during vertebrate germline development. • Dzip1 physically interacts with Dazl, which is essential for germline development. • Dzip1 is required for the development of Xenopus primordial germ cells. [ABSTRACT FROM AUTHOR]

Plants can survive in soils of low micromolar potassium (K+) concentrations. Root K+ intake is accomplished by the K+ channel AKT1 and KUP/HAK/KT type high-affinity K+ transporters. Arabidopsis HAK5 mutants impaired in low K+ acquisition have been identified already more than two decades ago, the molecular mechanism, however, is still a matter of debate also because of lack of direct measurements of HAK5-mediated K+ currents. When we expressed AtHAK5 in Xenopus oocytes together with CBL1/CIPK23, no inward currents were elicited in sufficient K+ media. Under low K+ and inward-directed proton motive force (PMF), the inward K+ current increased indicating that HAK5 energetically couples the uphill transport of K+ to the downhill flux of H+. At extracellular K+ concentrations above 25 μM, the initial rise in current was followed by a concentration-graded inactivation. When we replaced Tyr450 in AtHAK5 to Ala the K+ affinity strongly decreased, indicating that AtHAK5 position Y450 holds a key for K+ sensing and transport. When the soil K+ concentration drops toward the range that thermodynamically cannot be covered by AKT1, the AtHAK5 K+/H+ symporter progressively takes over K+ nutrition. Therefore, optimizing K+ use efficiency of crops, HAK5 could be key for low K+ tolerant agriculture. Plant survival under low K+ relies on the function of the K+ transporter HAK5. Here, the authors show the transport characteristics of Arabidopsis HAK5, unveiling its K+ and pH dependent activation and deactivation kinetics. [ABSTRACT FROM AUTHOR]

In inner ear hair cells, the stereocilia are inserted into a dense F‐actin‐enriched meshwork named the cuticular plate, which provides support to the stereocilia. Enah/Vasp‐like (EVL) was shown to localize at the cuticular plate, and evl knockdown leads to disrupted cuticular plate and disorganized stereocilia in Xenopus hair cells. In the present work, we show that Evl transcripts are specifically expressed in mouse hair cells, and EVL is localized to the cuticular plate. However, the cuticular plate and stereocilia are unaffected by Evl knockout, and auditory function is largely normal in Evl knockout mice. In conclusion, our present data suggest that EVL is not essential for cuticular plate and stereocilia development in mouse auditory hair cells. [ABSTRACT FROM AUTHOR]

High-affinity potassium transporters (HKTs) play an important role in plants responding to salt stress, but the transport properties of the soybean HKT transporters at the molecular level are still unclear. Here, using Xenopus oocyte as a heterologous expression system and two-electrode voltage-clamp technique, we identified four HKT transporters, GmHKT1;1, GmHKT1;2, GmHKT1;3 and GmHKT1;4, all of which belong to type I subfamily, but have distinct ion transport properties. While GmHKT1;1, GmHKT1;2 and GmHKT1;3 function as Na+ transporters, GmHKT1;1 is less selective against K+ than the two other transporters. Astonishingly, GmHKT1;4, which lacks transmembrane segments and has no ion permeability, is significantly expressed, and its gene expression pattern is different from the other three GmHKTs under salt stress. Interestingly, GmHKT1;4 reduced the Na+/K+ currents mediated by GmHKT1;1. Further study showed that the transport ability of GmHKT1;1 regulated by GmHKT1;4 was related to the structural differences in the first intracellular domain and the fourth repeat domain. Overall, we have identified one unique GmHKT member, GmHKT1;4, which modulates the Na+ and K+ transport ability of GmHKT1;1 via direct interaction. Thus, we have revealed a new type of HKT interaction model for altering their ion transport properties. [ABSTRACT FROM AUTHOR]

In certain tetraploid species resulting from interspecific hybridization, one parent's subgenome is known to selectively undergo DNA loss. The molecular mechanisms behind this remain unclear. In our study, we compared the genomes of a standard diploid species with two allotetraploid species from the Xenopus genus, both possessing L (longer) and S (shorter) homoeologous subgenomes. We observed substantial gene losses and intergenic DNA deletions in both the S and L subgenomes of the tetraploid species. Gene losses were around 1,000 to 3,000 for L and 4,000 to 6,000 for S, with especially prominent losses in the S subgenome. Many of these losses likely occurred shortly after interspecific hybridization in both L/S subgenomes. We also deduced frequent large inversions in the S subgenome. Upon reassessing transposon dynamics using updated genome databases, we reaffirmed heightened DNA transposon activity during the hybridization, as previously reported. We next investigated whether S subgenome-biased DNA loss could be correlated with the activation of DNA transposons following hybridization. Notably, distinct patterns were observed in the dynamics of DNA transposons between the L and S subgenomes. Several DNA transposon subfamilies correlated positively with DNA deletions in the S subgenome and negatively in the L subgenome. Based on these results, we propose a model that, upon and after hybridization between two related diploid Xenopus species, the mixture of their genomes resulted in the derepression of DNA transposons, especially in the S subgenome, leading to selective DNA loss in the S subgenome. [ABSTRACT FROM AUTHOR]

The African clawed frog (Xenopus laevis) endures prolonged periods of dehydration while estivating underground during the dry season. Epigenetic modifications play crucial roles in regulating gene expression in response to environmental changes. The elucidation of epigenetic changes relevant to survival could serve as a basis for further studies on organ preservation under extreme stress. The current study examined the relative protein levels of key enzymes involved in the arginine methylation of histones in the liver and kidney tissues of control versus dehydrated (35 ± 1%) X. laevis through immunoblotting. Protein arginine methyltransferases (PRMT) 4, 5, and 6 showed significant protein level decreases of 35 ± 3%, 71 ± 7%, and 25 ± 5%, respectively, in the liver tissues of the dehydrated frogs relative to controls. In contrast, PRMT7 exhibited an increase of 36 ± 4%. Similarly, the methylated histone markers H3R2m2a, H3R8m2a, and H3R8m2s were downregulated by 34 ± 11%, 15 ± 4%, and 42 ± 12%, respectively, in the livers of dehydrated frogs compared to controls. By contrast, the kidneys of dehydrated frogs showed an upregulation of histone markers. H3R2m2a, H3R8m2a, H3R8m2s, and H4R3m2a were significantly increased by 126 ± 12%, 112 ± 7%, 47 ± 13%, and 13 ± 3%, respectively. These changes can play vital roles in the metabolic reorganization of X. laevis during dehydration, and are likely to increase the chances of survival. In turn, the tissue-specific regulation of the histone arginine methylation mechanism suggests the importance of epigenetic regulation in the adaptation of X. laevis for whole-body dehydration. [ABSTRACT FROM AUTHOR]

Age-associated changes in DNA methylation have been characterized across various animals, but not yet in amphibians, which are of particular interest because they include widely studied model organisms. In this study, we present clear evidence that the aquatic vertebrate species Xenopus tropicalis displays patterns of age-associated changes in DNA methylation. We have generated whole-genome bisulfite sequencing (WGBS) profiles from skin samples of nine frogs representing young, mature, and old adults and characterized the gene- and chromosome-scale DNA methylation changes with age. Many of the methylation features and changes we observe are consistent with what is known in mammalian species, suggesting that the mechanism of age-related changes is conserved. Moreover, we selected a few thousand age-associated CpG sites to build an assay based on targeted DNA methylation analysis (TBSseq) to expand our findings in future studies involving larger cohorts of individuals. Preliminary results of a pilot TBSeq experiment recapitulate the findings obtained with WGBS setting the basis for the development of an epigenetic clock assay. The results of this study will allow us to leverage the unique resources available for Xenopus to study how DNA methylation relates to other hallmarks of ageing.

Abstract Background Regenerative medicine and transplantation science continuously seek methods to circumvent immune-mediated rejection and promote tissue regeneration. Sertoli cells, with their inherent immunoprotective properties, emerge as pivotal players in this quest. However, whether Sertoli cells can play immunomodulatory role in tadpole tail regeneration and can thus benefit the regeneration process are needed to be discovered. Methods Immature Sertoli cells from Xenopus tropicalis (XtiSCs) were transplanted into X. tropicalis tadpoles, followed by the amputation of the final third of their tails. We assessed the migration of XtiSCs, tail regeneration length, muscle degradation and growth, and macrophage counts across various regions including the entire tail, tail trunk, injection site, and regeneration site. The interactions between XtiSCs and macrophages were examined using a confocal microscope. To deplete macrophages, clodronate liposomes were administered prior to the transplantation of XtiSCs, while the administration of control liposomes acted as a negative control. Student’s t-test was used to compare the effects of XtiSCs injection to those of a 2/3PBS injection across groups with no liposomes, control liposomes, and clodronate liposomes. Results XtiSCs have excellent viability after transplantation to tadpole tail and remarkable homing capabilities to the regeneration site after tail amputation. XtiSCs injection increased macrophage numbers at 3 days post-amputation and 5 days post-amputation in the tail trunk, specifically at the injection site and at the regenerated tail, in a macrophage depleted environment (clodronate-liposome injection). What’s more, XtiSCs injection decreased muscle fibers degradation significantly at 1 day post-amputation and facilitated new muscle growth significantly at 3 days post-amputation. In addition, whole-mount immunostaining showed that some XtiSCs co-localized with macrophages. And we observed potential mitochondria transport from XtiSCs to macrophages using MitoTracker staining in tadpole tail. Conclusions Our study delineates the novel role of XtiSCs in facilitating muscle regeneration post tadpole tail amputation, underscoring a unique interaction with macrophages that is crucial for regenerative success. This study not only highlights the therapeutic potential of Sertoli cells in regenerative medicine but also opens avenues for clinical translation, offering insights into immunoregulatory strategies that could enhance tissue regeneration and transplant acceptance.

In all vertebrates, maintaining trunk posture primarily depends on descending commands originating from brainstem vestibulospinal nuclei. Despite being broadly outlined across species, the detailed anatomical and operational structure of these vestibulospinal networks remains poorly understood. Xenopus frogs have previously served as an excellent model for exploring such anatomical and functional aspects in relation to the animal’s behavioral requirements. In this study, we examined the reflex motor reactions induced by vestibular stimulation in pre-metamorphic tadpoles. Our findings indicate that natural vestibular stimulation in the horizontal plane yields greater efficacy compared to stimulation in other planes, a phenomenon replicated in a frequencydependent manner through specific galvanic stimulation (GVS) of the horizontal semicircular canals. With the exception of a very rostral cluster of neurons that receive vestibular inputs and project to the spinal cord, the overall anatomical segregation of vestibulospinal nuclei in the brainstem mirrors that observed in juvenile frogs. However, our results suggest closer similarities to mammalian organization than previously acknowledged. Moreover, we demonstrated that vestibulospinal cells project not only to spinal motoneurons in rostral segments but also to more distal segments that undergo regression during metamorphosis. Lastly, we illustrated how vestibular-induced spinal reflexes change during larval development, transitioning from tail swim-based activity to rostral trunk bursting responses, likely anticipating postural control in post-metamorphic frogs. [ABSTRACT FROM AUTHOR]

The TRESK (K2P18.1, KCNK18) background potassium channel is expressed in primary sensory neurons and has been reported to contribute to the regulation of pain sensations. In the present study, we examined the interaction of TRESK with NDFIP1 (Nedd4 family-interacting protein 1) in the Xenopus oocyte expression system by two-electrode voltage clamp and biochemical methods. We showed that the coexpression of NDFIP1 abolished the TRESK current under the condition where the other K+ channels were not affected. Mutations in the three PPxY motifs of NDFIP1, which are responsible for the interaction with the Nedd4 ubiquitin ligase, prevented a reduction in the TRESK current. Furthermore, the overexpression of a dominant-negative Nedd4 construct in the oocytes coexpressing TRESK with NDFIP1 partially reversed the down-modulating effect of the adaptor protein on the K+ current. The biochemical data were also consistent with the functional results. An interaction between epitope-tagged versions of TRESK and NDFIP1 was verified by co-immunoprecipitation experiments. The coexpression of NDFIP1 with TRESK induced the ubiquitination of the channel protein. Altogether, the results suggest that TRESK is directly controlled by and highly sensitive to the activation of the NDFIP1-Nedd4 system. The NDFIP1-mediated reduction in the TRESK component may induce depolarization, increase excitability, and attenuate the calcium dependence of the membrane potential by reducing the calcineurin-activated fraction in the ensemble background K+ current. [ABSTRACT FROM AUTHOR]

Retinal progenitor cells (RPCs) are a multipotent and highly proliferative population that give rise to all retinal cell types during organogenesis. Defining their molecular signature is a key step towards identifying suitable approaches to treat visual impairments. Here, we performed RNA sequencing of whole eyes from Xenopus at three embryonic stages and used differential expression analysis to define the transcriptomic profiles of optic tissues containing proliferating and differentiating RPCs during retinogenesis. Gene Ontology and KEGG pathway analyses showed that genes associated with developmental pathways (including Wnt and Hedgehog signaling) were upregulated during the period of active RPC proliferation in early retinal development (Nieuwkoop Faber st. 24 and 27). Developing eyes had dynamic expression profiles and shifted to enrichment for metabolic processes and phototransduction during RPC progeny specification and differentiation (st. 35). Furthermore, conserved adult eye regeneration genes were also expressed during early retinal development, including sox2, pax6, nrl, and Notch signaling components. The eye transcriptomic profiles presented here span RPC proliferation to retinogenesis and include regrowth-competent stages. Thus, our dataset provides a rich resource to uncover molecular regulators of RPC activity and will allow future studies to address regulators of RPC proliferation during eye repair and regrowth. [ABSTRACT FROM AUTHOR]

Na+/Cl− cotransporter 2 (Ncc2 or Slc12a10) is a membrane transport protein that belongs to the electroneutral cation-chloride cotransporter family. The Slc12a10 gene (slc12a10) is widely present in bony vertebrates but is deleted or pseudogenized in birds, some bony fishes, and most mammals. Slc12a10 is highly homologous to Ncc (Slc12a3 or Ncc1); however, there are only a few reports measuring the activity of Slc12a10. In this study, we focused on zebrafish Slc12a10.1 (zSlc12a10.1) and analyzed its activity using Xenopus oocyte electrophysiology. Analysis using Na+-selective microelectrodes showed that intracellular sodium activity (aNai) in zSlc12a10.1 oocytes was significantly decreased in Na+- or Cl−-free medium and recovered when Na+ or Cl− was readded to the medium. Similar analysis using a Cl−-selective microelectrode showed that intracellular chloride activity (aCli) in zSlc12a10.1 oocytes significantly decreased in Na+- or Cl−-free medium and recovered when Na+ or Cl− was readded to the medium. When a similar experiment was performed with a voltage clamp, the membrane current did not change when aNai of zSlc12a10.1 oocytes was decreased in Na+-free medium. Molecular phylogenetic and synteny analyses suggest that gene duplication between slc12a10.2 and slc12a10.3 in zebrafish is a relatively recent event, whereas gene duplication between slc12a10.1 and the ancestral gene of slc12a10.2/slc12a10.3 occurred at least about 2 million years ago. slc12a10 deficiency was observed in species belonging to Ictaluridae, Salmoniformes, Osmeriformes, Batrachoididae, Syngnathiformes, Gobiesociformes, Labriformes, and Tetraodontiformes. These results indicate that zebrafish Slc12a10.1 is an electroneutral Na+/Cl−cotransporter and establish its evolutionary position among various teleost slc12a10 paralogs. NEW & NOTEWORTHY: Na+/Cl− cotransporter 2 (Slc12a10; Ncc2) is a protein highly homologous to Ncc (Slc12a3; Ncc1); however, there are only a few reports measuring the activity of Slc12a10. Electrophysiological analysis of Xenopus oocytes expressing zebrafish Slc12a10.1 showed that Slc12a10.1 acts as an electroneutral Na+/Cl−cotransporter. This is the third report on the activity of Slc12a10, following previous reports on Slc12a10 in eels. [ABSTRACT FROM AUTHOR]

The signals that regulate peripheral blood vessel formation during development are still under investigation. The hormone leptin promotes blood vessel formation, adipose tissue establishment and expansion, tumor growth, and wound healing, but the underlying mechanisms for these actions are currently unknown. We investigated whether leptin promotes angiogenesis in the developing tail fin using embryonic transgenic xflk-1:GFP Xenopus laevis, which express a green fluorescent protein on vascular endothelial cells to mark blood vessels. We found that leptin protein is expressed in endothelial cells of developing blood vessels and that leptin treatment via injection increased phosphorylated STAT3 signaling, which is indicative of leptin activation of its receptor, in blood vessels of the larval tail fin. Leptin administration via media increased vessel length, branching, and reconnection with the cardinal vein, while decreased leptin signaling via immunoneutralization had an opposing effect on vessel development. We also observed disorganization of major vessels and microvessels of the tail fin and muscle when leptin signaling was decreased. Reduced leptin signaling lowered mRNA expression of cenpk , gpx1 , and mmp9 , markers for cell proliferation, antioxidation, and extracellular matrix remodeling/cell migration, respectively, in the developing tail, providing insight into three possible mechanisms underlying leptin's promotion of angiogenesis. Together these results illustrate that leptin levels are correlated with embryonic angiogenesis and that leptin coordinates multiple aspects of blood vessel growth and development, showing that leptin is an important morphogen during embryonic development. [Display omitted] • Leptin protein localizes and signals in blood vessels of Xenopus embryos. • Leptin increases multiple dimensions of blood vessel growth in the tadpole tail. • Decreased leptin signaling reduces vessel growth and disrupts organization. • Leptin signaling modulates expression of proliferation and ECM remodeling genes. • First evidence linking leptin and peripheral angiogenesis in vertebrate embryos. [ABSTRACT FROM AUTHOR]

Neural crest cells are a stem cell population unique to vertebrate embryos that retains broad multi-germ layer developmental potential through neurulation. Much remains to be learned about the genetic and epigenetic mechanisms that control the potency of neural crest cells. Here, we examine the role that epigenetic readers of the BET (bromodomain and extra terminal) family play in controlling the potential of pluripotent blastula and neural crest cells. We find that inhibiting BET activity leads to loss of pluripotency at blastula stages and a loss of neural crest at neurula stages. We compare the effects of HDAC (an eraser of acetylation marks) and BET (a reader of acetylation) inhibition and find that they lead to similar cellular outcomes through distinct effects on the transcriptome. Interestingly, loss of BET activity in cells undergoing lineage restriction is coupled to increased expression of genes linked to pluripotency and prolongs the competence of initially pluripotent cells to transit to a neural progenitor state. Together these findings advance our understanding of the epigenetic control of pluripotency and the formation of the vertebrate neural crest. [ABSTRACT FROM AUTHOR]

SoxB1 transcription factors (Sox2/3) are well known for their role in early neural fate specification in the embryo, but little is known about functional roles for SoxB1 factors in non-neural ectodermal cell types, such as the neural plate border (NPB). Using Xenopus laevis, we set out to determine whether SoxB1 transcription factors have a regulatory function in NPB formation. Here, we show that SoxB1 factors are necessary for NPB formation, and that prolonged SoxB1 factor activity blocks the transition from a NPB to a neural crest state. Using ChIP-seq, we demonstrate that Sox3 is enriched upstream of NPB genes in early NPB cells and in blastula stem cells. Depletion of SoxB1 factors in blastula stem cells results in downregulation of NPB genes. Finally, we identify Pou5f3 factors as potential Sox3 partners in regulating the formation of the NPB and show that their combined activity is needed for normal NPB gene expression. Together, these data identify a role for SoxB1 factors in the establishment and maintenance of the NPB, in part through partnership with Pou5f3 factors. [ABSTRACT FROM AUTHOR]

Xenopus embryos provide a favorable material to dissect the sequential steps that lead to dorsal-ventral (D-V) and anterior-posterior (A-P) cell differentiation. Here, we analyze the signaling pathways involved in this process using loss-of-function and gain-of-function approaches. The initial step was provided by Hwa, a transmembrane protein that robustly activates early β-catenin signaling when microinjected into the ventral side of the embryo leading to complete twinned axes. The following step was the activation of Xenopus Nodal-related growth factors, which could rescue the depletion of β-catenin and were themselves blocked by the extracellular Nodal antagonists Cerberus-Short and Lefty. During gastrulation, the Spemann-Mangold organizer secretes a cocktail of growth factor antagonists, of which the BMP antagonists Chordin and Noggin could rescue simultaneously D-V and A-P tissues in β-catenin-depleted embryos. Surprisingly, this rescue occurred in the absence of any β-catenin transcriptional activity as measured by β-catenin activated Luciferase reporters. The Wnt antagonist Dickkopf (Dkk1) strongly synergized with the early Hwa signal by inhibiting late Wnt signals. Depletion of Sizzled (Szl), an antagonist of the Tolloid chordinase, was epistatic over the Hwa and Dkk1 synergy. BMP4 mRNA injection blocked Hwa-induced ectopic axes, and Dkk1 inhibited BMP signaling late, but not early, during gastrulation. Several unexpected findings were made, e.g., well-patterned complete embryonic axes are induced by Chordin or Nodal in β-catenin knockdown embryos, dorsalization by Lithium chloride (LiCl) is mediated by Nodals, Dkk1 exerts its anteriorizing and dorsalizing effects by regulating late BMP signaling, and the Dkk1 phenotype requires Szl. [ABSTRACT FROM AUTHOR]

Self-regenerating trigger waves can spread rapidly through the crowded cytoplasm without diminishing in amplitude or speed, providing consistent, reliable, long-range communication. The macromolecular concentration of the cytoplasm varies in response to physiological and environmental fluctuations, raising the question of how or if trigger waves can robustly operate in the face of such fluctuations. Using Xenopus extracts, we find that mitotic and apoptotic trigger wave speeds are remarkably invariant. We derive a model that accounts for this robustness and for the eventual slowing at extremely high and low cytoplasmic concentrations. The model implies that the positive and negative effects of cytoplasmic concentration (increased reactant concentration vs. increased viscosity) are nearly precisely balanced. Accordingly, artificially maintaining a constant cytoplasmic viscosity during dilution abrogates this robustness. The robustness in trigger wave speeds may contribute to the reliability of the extremely rapid embryonic cell cycle. Mitosis and apoptosis spread as trigger waves through the Xenopus cytoplasm. The present work shows that wave speed is robust to cytoplasmic concentration and dilution, thanks to a near-exact balance of diffusion, concentration, and crowding effects. [ABSTRACT FROM AUTHOR]

This paper introduces a single-cell atlas for pivotal developmental stages in Xenopus , encompassing gastrulation, neurulation, and early tailbud. Notably surpassing its predecessors, the new atlas enhances gene mapping, read counts, and gene/cell type nomenclature. Leveraging the latest Xenopus tropic alis genome version, alongside advanced alignment pipelines and machine learning for cell type assignment, this release maintains consistency with previous cell type annotations while rectifying nomenclature issues. Employing an unbiased approach for cell type assignment proves especially apt for embryonic contexts, given the considerable number of non-terminally differentiated cell types. An alternative cell type attribution here adopts a fuzzy, non-deterministic stance, capturing the transient nature of early embryo progenitor cells by presenting an ensemble of types in superposition. The value of the new resource is emphasized through numerous examples, with a focus on previously unexplored germ cell populations where we uncover novel transcription onset features. Offering interactive exploration via a user-friendly web portal and facilitating complete data downloads, this atlas serves as a comprehensive and accessible reference. [Display omitted] [ABSTRACT FROM AUTHOR]

Human pigmentary disorders encompass a broad spectrum of phenotypic changes arising from disruptions in various stages of melanocyte formation, the melanogenesis process, or the transfer of pigment from melanocytes to keratinocytes. A large number of pigmentation genes associated with pigmentary disorders have been identified, many of them awaiting in vivo confirmation. A more comprehensive understanding of the molecular basis of pigmentary disorders requires a vertebrate animal model where changes in pigmentation are easily observable in vivo and can be combined to genomic modifications and gain/loss‐of‐function tools. Here we present the amphibian Xenopus with its unique features that fulfill these requirements. Changes in pigmentation are particularly easy to score in Xenopus embryos, allowing whole‐organism based phenotypic screening. The development and behavior of Xenopus melanocytes closely mimic those observed in mammals. Interestingly, both Xenopus and mammalian skins exhibit comparable reactions to ultraviolet radiation. This review highlights how Xenopus constitutes an alternative and complementary model to the more commonly used mouse and zebrafish, contributing to the advancement of knowledge in melanocyte cell biology and related diseases. [ABSTRACT FROM AUTHOR]

Solute carrier family 26 (Slc26) is a family of anion exchangers with 11 members in mammals (named Slc26a1‐a11). Here, we identified a novel member of the slc26 family, slc26a12, located in tandem with slc26a2 in the genomes of several vertebrate lineages. BLAST and synteny analyses of various jawed vertebrate genome databases revealed that slc26a12 is present in coelacanths, amphibians, reptiles, and birds but not in cartilaginous fishes, lungfish, mammals, or ray‐finned fishes. In some avian and reptilian lineages such as owls, penguins, egrets, and ducks, and most turtles examined, slc26a12 was lost or pseudogenized. Phylogenetic analysis showed that Slc26a12 formed an independent branch with the other Slc26 members and Slc26a12, Slc26a1 and Slc26a2 formed a single branch, suggesting that these three members formed a subfamily in Slc26. In jawless fish, hagfish have two genes homologous to slc26a2 and slc26a12, whereas lamprey has a single gene homologous to slc26a2. African clawed frogs express slc26a12 in larval gills, skin, and fins. These results show that slc26a12 was present at least before the separation of lobe‐finned fish and tetrapods; the name slc26a12 is appropriate because the gene duplication occurred in the distant past. [ABSTRACT FROM AUTHOR]

During the formation of the neural tube, the primordium of the vertebrate central nervous system, the actomyosin activity of cells in different regions drives neural plate bending. However, how the stiffness of the neural plate and surrounding tissues is regulated and mechanically influences neural plate bending has not been elucidated. Here, we used atomic force microscopy to reveal the relationship between the stiffness of the neural plate and the mesoderm during Xenopus neural tube formation. Measurements with intact embryos revealed that the stiffness of the neural plate was consistently higher compared with the non‐neural ectoderm and that it increased in an actomyosin activity‐dependent manner during neural plate bending. Interestingly, measurements of isolated tissue explants also revealed that the relationship between the stiffness of the apical and basal sides of the neural plate was reversed during bending and that the stiffness of the mesoderm was lower than that of the basal side of the neural plate. The experimental elevation of mesoderm stiffness delayed neural plate bending, suggesting that low mesoderm stiffness mechanically supports neural tube closure. This study provides an example of mechanical interactions between tissues during large‐scale morphogenetic movements. [ABSTRACT FROM AUTHOR]

Mycobacterium abscessus (Mab) is an emerging human pathogen that has a high rate of incidence in immunocompromised individuals. We have found a putative secondary metabolite pathway within Mab, which may be a key factor in its pathogenesis. This novel pathway is encoded in a gene cluster spanning MAB_0284c to 0305 and is related to Streptomyces pathways, producing the secondary metabolites streptonigrin and nybomycin. We constructed an in-frame deletion of the MAB_0295 (phzC) gene and tested it in our Xenopus laevis animal model. We have previously shown that X. laevis tadpoles, which have functional lungs and T cells, can serve as a reliable comparative model for persistent Mab infection and pathogenesis. Here, we report that tadpoles intraperitoneally infected with the ∆phzC mutant exhibit early decreased bacterial loads and significantly increased survival compared with those infected with WT Mab. ∆phzC mutant Mab also induced lower transcript levels of several pro-inflammatory cytokines (IL-1β, TNF-α, iNOS, IFN-γ) than those of WT Mab in the liver and lungs. In addition, there was impaired macrophage recruitment and decreased macrophage infection in tadpoles infected with the ∆phzC mutant, by tail wound inoculation, compared to those infected with the WT bacteria, as assayed by intravital confocal microscopy. These data underline the relevance and usefulness of X. laevis tadpoles as a novel comparative animal model to identify genetic determinants of Mab immunopathogenesis, suggesting a role for this novel and uncharacterized pathway in Mab pathogenesis and macrophage recruitment. [ABSTRACT FROM AUTHOR]

Although polyploidization is frequent in development, cancer, and evolution, impacts on animal metabolism are poorly understood. In Xenopus frogs, the number of genome copies (ploidy) varies across species and can be manipulated within a species. Here, we show that triploid tadpoles contain fewer, larger cells than diploids and consume oxygen at a lower rate. Drug treatments revealed that the major processes accounting for tadpole energy expenditure include cell proliferation, biosynthesis, and maintenance of plasma membrane potential. While inhibiting cell proliferation did not abolish the oxygen consumption difference between diploids and triploids, treatments that altered cellular biosynthesis or electrical potential did. Combining these results with a simple mathematical framework, we propose that the decrease in total cell surface area lowered production and activity of plasma membrane components including the Na+/K+ ATPase, reducing energy consumption in triploids. Comparison of Xenopus species that evolved through polyploidization revealed that metabolic differences emerged during development when cell size scaled with genome size. Thus, ploidy affects metabolism by altering the cell surface area to volume ratio in a multicellular organism.

Retinal ganglion cell (RGC) axons of the African clawed frog, Xenopus laevis, unlike those of mammals, are capable of regeneration and functional reinnervation of central brain targets following injury. Here, we describe a tadpole optic nerve crush (ONC) procedure and assessments of brain reinnervation based on live imaging of RGC-specific transgenes which, when paired with CRISPR/Cas9 injections at the one-cell stage, can be used to assess the function of regeneration-associated genes in vivo in F0 animals. Using this assay, we find that map3k12, also known as dual leucine zipper kinase (Dlk), is necessary for RGC axonal regeneration and acts in a dose-dependent manner. Loss of Dlk does not affect RGC innervation of the brain during development or visually driven behavior but does block both axonal regeneration and functional vision restoration after ONC. Dlk loss does not alter the acute changes in mitochondrial movement that occur within RGC axons hours after ONC but does completely block the phosphorylation and nuclear translocation of the transcription factor Jun within RGCs days after ONC; yet, Jun is dispensable for reinnervation. These results demonstrate that in a species fully capable of regenerating its RGC axons, Dlk is essential for the axonal injury signal to reach the nucleus but may affect regeneration through a different pathway than by which it signals in mammalian RGCs.

During the rapid and reductive cleavage divisions of early embryogenesis, subcellular structures such as the nucleus and mitotic spindle scale to decreasing cell size. Mitotic chromosomes also decrease in size during development, presumably to scale coordinately with mitotic spindles, but the underlying mechanisms are unclear. Here we combine in vivo and in vitro approaches using eggs and embryos from the frog Xenopus laevis to show that mitotic chromosome scaling is mechanistically distinct from other forms of subcellular scaling. We found that mitotic chromosomes scale continuously with cell, spindle, and nuclear size in vivo. However, unlike for spindles and nuclei, mitotic chromosome size cannot be reset by cytoplasmic factors from earlier developmental stages. In vitro, increasing nuclear-cytoplasmic (N/C) ratio is sufficient to recapitulate mitotic chromosome scaling, but not nuclear or spindle scaling, through differential loading of maternal factors during interphase. An additional pathway involving importin α scales mitotic chromosomes to cell surface area/volume ratio (SA/V) during metaphase. Finally, single-chromosome immunofluorescence and Hi-C data suggest that mitotic chromosomes shrink during embryogenesis through decreased recruitment of condensin I, resulting in major rearrangements of DNA loop architecture to accommodate the same amount of DNA on a shorter chromosome axis. Together, our findings demonstrate how mitotic chromosome size is set by spatially and temporally distinct developmental cues in the early embryo.

Genome and cell size are strongly correlated across species1,2,3,4,5,6 and influence physiological traits like developmental rate.7,8,9,10,11,12 Although size scaling features such as the nuclear-cytoplasmic (N/C) ratio are precisely maintained in adult tissues,13 it is unclear when during embryonic development size scaling relationships are established. Frogs of the genus Xenopus provide a model to investigate this question, since 29 extant Xenopus species vary in ploidy from 2 to 12 copies (N) of the ancestral frog genome, ranging from 20 to 108 chromosomes.14,15 The most widely studied species, X. laevis (4N = 36) and X. tropicalis (2N = 20), scale at all levels, from body size to cellular and subcellular levels.16 Paradoxically, the rare, critically endangered dodecaploid (12N = 108) Xenopus longipes (X. longipes) is a small frog.15,17 We observed that despite some morphological differences, X. longipes and X. laevis embryogenesis occurred with similar timing, with genome to cell size scaling emerging at the swimming tadpole stage. Across the three species, cell size was determined primarily by egg size, whereas nuclear size correlated with genome size during embryogenesis, resulting in different N/C ratios in blastulae prior to gastrulation. At the subcellular level, nuclear size correlated more strongly with genome size, whereas mitotic spindle size scaled with cell size. Our cross-species study indicates that scaling of cell size to ploidy is not due to abrupt changes in cell division timing, that different size scaling regimes occur during embryogenesis, and that the developmental program of Xenopus is remarkably consistent across a wide range of genome and egg sizes.

Activation of the Wnt pathway lies at the core of many human cancers. Wnt and macropinocytosis are often active in the same processes, and understanding how Wnt signaling and membrane trafficking cooperate should improve our understanding of embryonic development and cancer. Here, we show that a macropinocytosis activator, the tumor promoter phorbol 12-myristate 13-acetate (PMA), enhances Wnt signaling. Experiments using the Xenopus embryo as an in vivo model showed marked cooperation between the PMA phorbol ester and Wnt signaling, which was blocked by inhibitors of macropinocytosis, Rac1 activity, and lysosome acidification. Human colorectal cancer tissue arrays and xenografts in mice showed a correlation of cancer progression with increased macropinocytosis/multivesicular body/lysosome markers and decreased GSK3 levels. The crosstalk between canonical Wnt, focal adhesions, lysosomes, and macropinocytosis suggests possible therapeutic targets for cancer progression in Wnt-driven cancers.

Histone acetylation is a pivotal epigenetic modification that controls chromatin structure and regulates gene expression. It plays an essential role in modulating zygotic transcription and cell lineage specification of developing embryos. While the outcomes of many inductive signals have been described to require enzymatic activities of histone acetyltransferases and deacetylases (HDACs), the mechanisms by which HDACs confine the utilization of the zygotic genome remain to be elucidated. Here, we show that histone deacetylase 1 (Hdac1) progressively binds to the zygotic genome from mid-blastula and onward. The recruitment of Hdac1 to the genome at blastula is instructed maternally. Cis-regulatory modules (CRMs) bound by Hdac1 possess epigenetic signatures underlying distinct functions. We highlight a dual function model of Hdac1 where Hdac1 not only represses gene expression by sustaining a histone hypoacetylation state on inactive chromatin, but also maintains gene expression through participating in dynamic histone acetylation-deacetylation cycles on active chromatin. As a result, Hdac1 maintains differential histone acetylation states of bound CRMs between different germ layers and reinforces the transcriptional program underlying cell lineage identities, both in time and space. Taken together, our study reveals a comprehensive role for Hdac1 during early vertebrate embryogenesis.

Protocadherin19 (PCDH19)-related epilepsy syndrome is a rare disorder characterized by early-onset epilepsy, intellectual disability, and autistic behaviors. PCDH19 is located on the X chromosome and encodes a calcium-dependent single-pass transmembrane protein, which regulates cell-to-cell adhesion through homophilic binding. In human, 90% of heterozygous females, containing PCDH19 wild-type and mutant cells due to random X inactivation, are affected, whereas mutant males, containing only mutant cells, are typically not. The current view, the cellular interference, is that the altered interactions between wild-type and mutant cells during development, rather than loss of function itself, are responsible. However, studies using Pcdh19 knockout mice showed that the complete loss of function also causes autism-like behaviors both in males and females, suggesting that other functions of PCDH19 may also contribute to pathogenesis. To address whether mosaicism is required for PCDH19-related epilepsy, we generated Xenopus tropicalis tadpoles with complete or mosaic loss of function by injecting antisense morpholino oligonucleotides into the blastomeres of neural lineage at different stages of development. We found that either mosaic or complete knockdown results in seizure-like behaviors, which could be rescued by antiseizure medication, and repetitive behaviors. Our results suggest that the loss of PCDH19 function itself, in addition to cellular interference, may also contribute to PCDH19-related epilepsy. [ABSTRACT FROM AUTHOR]

Invasive species threaten biodiversity globally. Amphibians are one of the most threatened vertebrate taxa and are particularly sensitive to invasive species, including other amphibians. African clawed frogs (Xenopus laevis) are native to Southern Africa but have subsequently become invasive on multiple continents—including multiple parts of North America—due to releases from the pet and biomedical trades. Despite their prevalence as a global invader, the impact of X. laevis remains understudied. This includes the Pacific Northwest of the USA, which now hosts multiple expanding X. laevis populations. For many amphibians, chemical cues communicate important information, including the presence of predators. Here, we tested the role chemical cues may play in mediating interactions between feral X. laevis and native amphibians in the Pacific Northwest. We tested whether native red-legged frog (Rana aurora) tadpoles display an antipredator response to non-native frog (X. laevis) or native newt (rough-skinned newts, Taricha granulosa) predator chemical stimuli. We found that R. aurora tadpoles exhibited pronounced anti-predator responses when exposed to chemical cues from T. granulosa but did not display anti-predator response to invasive X. laevis chemical cues. We also began experimentally testing whether T. granulosa—which produce a powerful neurotoxin tetrodotoxin (TTX)—may elicit an anti-predator response in X. laevis, that could serve to deter co-occupation. However, our short-duration experiments found that X. laevis were attracted to newt chemical stimuli rather than deterred. Our findings show that X. laevis likely poses a threat to native amphibians, and that these native species may also be particularly vulnerable to this invasive predator, compared to native predators, because toxic native newts may not limit X. laevis invasions. Our research provides some of the first indications that native Pacific Northwest species may be threatened by feral X. laevis and provides a foundation for future experiments testing potential management techniques for X. laevis. [ABSTRACT FROM AUTHOR]

Ionizing radiation (IR) causes DNA damage, particularly DNA double-strand breaks (DSBs), which have significant implications for genome stability. The major pathways of repairing DSBs are homologous recombination (HR) and nonhomologous end joining (NHEJ). However, the repair mechanism of IR-induced DSBs in embryos is not well understood, despite extensive research in somatic cells. The externally developing aquatic organism, Xenopus tropicalis , serves as a valuable model for studying embryo development. A significant increase in zygotic transcription occurs at the midblastula transition (MBT), resulting in a longer cell cycle and asynchronous cell divisions. This study examines the impact of X-ray irradiation on Xenopus embryos before and after the MBT. The findings reveal a heightened X-ray sensitivity in embryos prior to the MBT, indicating a distinct shift in the DNA repair pathway during embryo development. Importantly, we show a transition in the dominant DSB repair pathway from NHEJ to HR before and after the MBT. These results suggest that the MBT plays a crucial role in altering DSB repair mechanisms, thereby influencing the IR sensitivity of developing embryos. [ABSTRACT FROM AUTHOR]

Hearing in infants is essential for brain development, acquisition of verbal language skills, and development of social interactions. Therefore, it is important to diagnose hearing loss soon after birth so that interventions can be provided as early as possible. Most newborns in the United States are screened for hearing deficits and commercially available next‐generation sequencing hearing loss panels often can identify the causative gene, which may also identify congenital defects in other organs. One of the most prevalent autosomal dominant congenital hearing loss syndromes is branchio‐oto‐renal syndrome (BOR), which also presents with defects in craniofacial structures and the kidney. Currently, mutations in three genes, SIX1, SIX5, and EYA1, are known to be causative in about half of the BOR patients that have been tested. To uncover new candidate genes that could be added to congenital hearing loss genetic screens, we have combined the power of Drosophila mutants and protein biochemical assays with the embryological advantages of Xenopus, a key aquatic animal model with a high level of genomic similarity to human, to identify potential Six1 transcriptional targets and interacting proteins that play a role during otic development. We review our transcriptomic, yeast 2‐hybrid, and proteomic approaches that have revealed a large number of new candidates. We also discuss how we have begun to identify how Six1 and co‐factors interact to direct developmental events necessary for normal otic development. Research Highlights: Branchio‐oto‐renal patients have hearing loss and kidney defects.Mutations in SIX1, SIX5, and EYA1 are causative in about half of cases.To uncover putative new candidate genes, we used Xenopus and identified novel transcriptional targets and interacting proteins. [ABSTRACT FROM AUTHOR]

Traditionally, the Amphibian Metamorphosis Assay (AMA; OECD TG 231) is performed by exposing Xenopus laevis tadpoles to test substances dissolved in laboratory water. Recently, the use of dietary administration has been proposed to combat poorly soluble test substances in ecotoxicologically‐based regulatory endocrine disruption (ED) studies, specifically the AMA warranting an investigation into the efficacy of dietary administration. An efficacy study comprised of two phases: 1) evaluation of the physical influence of the loading process via solvent and 10, 1, and 0.1 mg/l test substance or surrogate (sunflower oil, SFO) on the Sera® Micron Nature (SMN) diet, and 2) performance of a modified AMA in which Nieuwkoop and Faber (NF) stage 51 X. laevis larvae were exposed to dechlorinated tap water using one concentration of the SFO in the diet for 21 days, was performed. In phase 1, the addition of acetone or acetone with bis(2‐propylheptyl) phthalate (DPHP) or SFO to SMN with subsequent solvent purge altered the diet reducing the density of the liquified diet and dietary pellet size following centrifugation indicative of alteration of the physical properties of the diet. Treatments used in the modified AMA were acetone alone and 0.1 mg/l SFO dissolved in acetone. These treatments were evaluated against an SMN benchmark using standard AMA endpoints. Both the acetone‐treated SMN and 0.1 mg/l SFO‐treated diets significantly reduced survival rates, 67 and 70% relative to the SMN benchmark (100%), decreased developmental stage distribution and snout‐vent length‐normalized hind limb length relative to the SMN benchmark, and slightly increased the prevalence and severity of thyroid follicular cell hypertrophy. Although the acetone‐treated diets may have impacted the hypothalamo‐pituitary‐thyroid axis, clinical signs of gastrointestinal impaction and tail flexure were also observed in the acetone‐treated diets, but not the SMN diet alone. Ultimately, test substance exposure via the diet in an AMA study can produce results that may confound data interpretation, which suggests that the traditional aqueous exposure route is generally more appropriate. Dietary administration has been proposed to combat poorly soluble test substances in ecotoxicologically based regulatory endocrine disruption (ED) studies warranting an investigation into the efficacy of dietary administration. Acetone‐treated diets significantly reduced survival rates, decreased developmental stage distribution and snout‐vent length‐normalized hind limb length, and slightly increased the prevalence and severity of thyroid follicular cell hypertrophy. Test substance exposure via the diet in an AMA study can confound data interpretation, and traditional aqueous exposure route is generally more appropriate. [ABSTRACT FROM AUTHOR]

In Asian honeybees, virgin queens typically only mate during a single nuptial flight before founding a colony. This behavior is controlled by the queen-released mandibular pheromone (QMP). 9-oxo-(E)-2-decenoic acid (9-ODA), a key QMP component, acts as sex pheromone and attracts drones. However, how the queens prevent additional mating remains elusive. Here, we show that the secondary QMP component methyl p-hydroxybenzoate (HOB) released by mated queens inhibits male attraction to 9-ODA. Results from electrophysiology and in situ hybridization assay indicated that HOB alone significantly reduces the spontaneous spike activity of 9-ODA-sensitive neurons, and AcerOr11 is specifically expressed in sensilla placodea from the drone's antennae, which are the sensilla that narrowly respond to both 9-ODA and HOB. Deorphanization of AcerOr11 in Xenopus oocyte system showed 9-ODA induces robust inward (regular) currents, while HOB induces inverse currents in a dose-dependent manner. This suggests that HOB potentially acts as an inverse agonist against AcerOr11. Identifying HOB, a substance of the queen mandibular pheromone, as an anti-aphrodisiac in Asian honeybee chemical communication. [ABSTRACT FROM AUTHOR]

Conditionally controlled antisense oligonucleotides provide precise interrogation of gene function at different developmental stages in animal models. Only one example of small molecule‐induced activation of antisense function exist. This has been restricted to cyclic caged morpholinos that, based on sequence, can have significant background activity in the absence of the trigger. Here, we provide a new approach using azido‐caged nucleobases that are site‐specifically introduced into antisense morpholinos. The caging group design is a simple azidomethylene (Azm) group that, despite its very small size, efficiently blocks Watson–Crick base pairing in a programmable fashion. Furthermore, it undergoes facile decaging via Staudinger reduction when exposed to a small molecule phosphine, generating the native antisense oligonucleotide under conditions compatible with biological environments. We demonstrated small molecule‐induced gene knockdown in mammalian cells, zebrafish embryos, and frog embryos. We validated the general applicability of this approach by targeting three different genes. [ABSTRACT FROM AUTHOR]

During organ regeneration, after the initial responses to injury, gene expression patterns similar to those in normal development are reestablished during subsequent morphogenesis phases. This supports the idea that regeneration recapitulates development and predicts the existence of genes that reboot the developmental program after the initial responses. However, such rebooting mechanisms are largely unknown. Here, we explore core rebooting factors that operate during Xenopus limb regeneration. Transcriptomic analysis of larval limb blastema reveals that hoxc12/c13 show the highest regeneration specificity in expression. Knocking out each of them through genome editing inhibits cell proliferation and expression of a group of genes that are essential for development, resulting in autopod regeneration failure, while limb development and initial blastema formation are not affected. Furthermore, the induction of hoxc12/c13 expression partially restores froglet regenerative capacity which is normally very limited compared to larval regeneration. Thus, we demonstrate the existence of genes that have a profound impact alone on rebooting of the developmental program in a regeneration-specific manner. During organ regeneration, gene expression patterns similar to those in normal development are reestablished. Here, Kawasumi-Kita et al. explore core rebooting factors that operate during Xenopus limb regeneration. Their results indicate that hoxc12 and hoxc13 are critical for reactivating tissue growth. [ABSTRACT FROM AUTHOR]