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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.

Direct discharge of aquaculture wastewater may have toxic effects, due to the presence of heavy metals, antibiotics, and even resistant pathogens, but little attention has been given. Here, tanks simulating a wild ecosystem were built to study the effects of long-term exposure to duck wastewater containing oxytetracycline (OTC) and/or arsenic (As) on the growth, physiological function, and gut microbiota evolution of Xenopus tropicalis. The results showed that duck wastewater had no apparent impact on X. tropicalis, but the impact increased significantly (P0.05) with exposure to OTC and/or As, especially the impact on body weight and growth rate. Biochemical indicators revealed varying degrees of oxidative stress damage, hepatotoxicity (inflammation, necrosis, and sinusoids), and collagen fibrosis of X. tropicalis in all treated groups after 72 days of exposure, which indirectly inhibited X. tropicalis growth. Moreover, 16S rDNA amplicon sequencing results showed that the gut microbiota structure and metabolic function were perturbed after chronic exposure, which might be the leading cause of growth inhibition. Interestingly, the abundance of intestinal resistance genes (RGs) increased with exposure time owing to the combined direct and indirect effects of stress factors in duck wastewater. Moreover, once the RGs were expressed, the resistance persisted for at least 24 days, especially that conferred by tetA. These results provide evidence of the toxic effects of DW containing OTC (0.1-4.0 mg/L) and/or As (0.3-3.5 µg/L) on amphibians and indicate that it is vital to limit the usage of heavy metals and antibiotics on farms to control the biotoxicity of wastewater.

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.

Cancer is a multifactorial disease that constitutes a serious public health problem worldwide. Prostate cancer advanced stages are associated with the development of androgen-independent tumors and an apoptosis-resistant phenotype that progresses to metastasis. By studying androgen-independent lymphoid nodule carcinoma of the prostate (LNCaP) cells induced to apoptosis by serum elimination, we identified the activation of a non-selective cationic channel of 23pS conductance that promotes incoming Ca2+ currents, as well as apoptosis final stages. arp2cDNA was isolated and identified to be of the same cell type, and mRNA was expressed in Xenopus laevis oocytes, which was found to be associated with the activation of incoming Ca2+ currents and induction to apoptosis. cDNA, which encodes the ARP2 protein, was overexpressed in LNCaP cells and Chinese hamster ovary cells, which induced apoptosis. Our evidence suggests that protein ARP2 overexpression and transit to the cell membrane allows an increased Ca2+ incoming current that initiates the apoptosis process in epithelial-type cells whose phenotype shows resistance to programmed cell death.

Rice has a large number of nitrate or peptide transporter family (NPF) genes, but the effects of most members on rice growth and development are unknown. We report that OsNPF5.16, a nitrate transporter gene with natural variation in its promoter sequence, is essential for rice growth and yield. The promoter sequence showed various differences between indica and japonica cultivars, and higher expression of OsNPF5.16 was found in indica cultivars with higher plant weight and more tillers than japonica cultivars. OsNPF5.16 was highly expressed in roots, tiller basal parts, and leaf sheaths, and its protein was localized on the plasma membrane. In cRNA-injected Xenopus laevis oocytes, OsNPF5.16 transport of nitrate at high nitrate concentration depended on pH. Overexpression of OsNPF5.16 increased nitrate content and total nitrogen content in leaf sheath as well as biomass and tiller bud length in rice. Elevated expression of OsNPF5.16 increased rice tiller number and grain yield by regulating cytokinin levels. Inhibition of OsNPF5.16 expression showed the opposite effects. Regulating OsNPF5.16 expression has potential for improving rice grain yield.

Regeneration of complex tissues is initiated by an injury-induced stress response, eventually leading to activation of developmental signaling pathways such as Wnt signaling. How early injury cues are interpreted and coupled to activation of these developmental signals and their targets is not well understood. Here, we show that Hif1α, a stress induced transcription factor, is required for tail regeneration in Xenopus tropicalis. We find that Hif1α is required for regeneration of differentiated axial tissues, including axons and muscle. Using RNA-sequencing, we find that Hif1α and Wnt converge on a broad set of genes required for posterior specification and differentiation, including the posterior hox genes. We further show that Hif1α is required for transcription via a Wnt-responsive element, a function that is conserved in both regeneration and early neural patterning. Our findings indicate that Hif1α has regulatory roles in Wnt target gene expression across multiple tissue contexts.

During metazoan early embryogenesis, the intracellular properties of proteins and organelles change dynamically through rapid cleavage. In particular, a change in the nucleus size is known to contribute to embryonic development-dependent cell cycle and gene expression regulation. Here, we compared the nuclear sizes of various blastomeres from developing Xenopus embryos and analyzed the mechanisms that control the nuclear expansion dynamics by manipulating the amount of intracellular components in a cell-free system. There was slower nuclear expansion during longer interphase durations in blastomeres from vegetal hemispheres than those from animal hemispheres. Furthermore, upon recapitulating interphase events by manipulating the concentration of yolk platelets, which are originally rich in the vegetal blastomeres, in cell-free cytoplasmic extracts, there was slower nuclear expansion and DNA replication as compared to normal yolk-free conditions. Under these conditions, the supplemented yolk platelets accumulated around the nucleus in a microtubule-dependent manner and impeded organization of the endoplasmic reticulum network. Overall, we propose that yolk platelets around the nucleus reduce membrane supply from the endoplasmic reticulum to the nucleus, resulting in slower nuclear expansion in the yolk-rich vegetal blastomeres.

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.

The Rad, Rem, Rem2, and Gem/Kir (RGK) sub-family of small GTP-binding proteins are crucial in regulating high voltage-activated (HVA) calcium channels. RGK proteins inhibit calcium current by either promoting endocytosis or reducing channel activity. They all can associate directly with Ca2+ channel β subunit (CaVβ), and the binding between CaVα1/CaVβ appears essential for the endocytic promotion of CaV1.X, CaV2.1, and CaV2.2 channels. In this study, we investigated the inhibition of CaV2.3 channels by RGK proteins in the absence of CaVβ. To this end, Xenopus laevis oocytes expressing CaV2.3 channels devoid of auxiliary subunit were injected with purified Gem and Rem and found that only Gem had an effect. Ca currents and charge movements were reduced by injection of Gem, pointing to a reduction in the number of channels in the plasma membrane. Since this reduction was ablated by co-expression of the dominant-negative mutant of dynamin K44A, enhanced endocytosis appears to mediate this reduction in the number of channels. Thus, Gem inhibition of CaV2.3 channels would be the only example of a CaVβ independent promotion of dynamin-dependent endocytosis.

The ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) is essential for the control of mitosis, and its activity is subject to tight regulation. In early mitosis, APC/C is inhibited by the mitotic checkpoint system, but subsequently it regains activity and promotes metaphase-anaphase transition by targeting cyclin B and securin for degradation. The phosphorylation of APC/C by the mitotic protein kinase Cdk1-cyclin B facilitates its interaction with its coactivator Cdc20, while the phosphorylation of Cdc20 inhibits its binding to APC/C. This raises the question of how Cdc20 binds to APC/C under conditions of high Cdk1 activity. It seemed possible that the opposing action of protein phosphatases produces a fraction of unphosphorylated Cdc20 that binds to APC/C. We found, however, that while inhibitors of protein phosphatases PP2A and PP1 increased the overall phosphorylation of Cdc20 in anaphase extracts from Xenopus eggs, they did not decrease the levels of Cdc20 bound to APC/C. Searching for alternative mechanisms, we found that following the binding of Cdc20 to APC/C, it became significantly protected against phosphorylation by Cdk1. Protection was mainly at threonine sites at the N-terminal region of Cdc20, known to affect its interaction with APC/C. A model is proposed according to which a pool of unphosphorylated Cdc20, originating from initial stages of mitosis or from phosphatase action, combines with phosphorylated APC/C and thus becomes stabilized against further phosphorylation, possibly by steric hindrance of Cdk1 action. This pool of APC Cdc20 appears to be required for the regulation of APC/C activity at different stages of mitosis.

Microphthalmia is a rare developmental eye disorder affecting 1 in 7000 births. It is defined as a small (axial length ⩾2 standard deviations below the age-adjusted mean) underdeveloped eye, caused by disruption of ocular development through genetic or environmental factors in the first trimester of pregnancy. Clinical phenotypic heterogeneity exists amongst patients with varying levels of severity, and associated ocular and systemic features. Up to 11% of blind children are reported to have microphthalmia, yet currently no treatments are available. By identifying the aetiology of microphthalmia and understanding how the mechanisms of eye development are disrupted, we can gain a better understanding of the pathogenesis. Animal models, mainly mouse, zebrafish and Xenopus, have provided extensive information on the genetic regulation of oculogenesis, and how perturbation of these pathways leads to microphthalmia. However, differences exist between species, hence cellular models, such as patient-derived induced pluripotent stem cell (iPSC) optic vesicles, are now being used to provide greater insights into the human disease process. Progress in 3D cellular modelling techniques has enhanced the ability of researchers to study interactions of different cell types during eye development. Through improved molecular knowledge of microphthalmia, preventative or postnatal therapies may be developed, together with establishing genotype–phenotype correlations in order to provide patients with the appropriate prognosis, multidisciplinary care and informed genetic counselling. This review summarises some key discoveries from animal and cellular models of microphthalmia and discusses how innovative new models can be used to further our understanding in the future.Plain language summaryAnimal and Cellular Models of the Eye Disorder, Microphthalmia (Small Eye) Microphthalmia, meaning a small, underdeveloped eye, is a rare disorder that children are born with. Genetic changes or variations in the environment during the first 3 months of pregnancy can disrupt early development of the eye, resulting in microphthalmia. Up to 11% of blind children have microphthalmia, yet currently no treatments are available. By understanding the genes necessary for eye development, we can determine how disruption by genetic changes or environmental factors can cause this condition. This helps us understand why microphthalmia occurs, and ensure patients are provided with the appropriate clinical care and genetic counselling advice. Additionally, by understanding the causes of microphthalmia, researchers can develop treatments to prevent or reduce the severity of this condition. Animal models, particularly mice, zebrafish and frogs, which can also develop small eyes due to the same genetic/environmental changes, have helped us understand the genes which are important for eye development and can cause birth eye defects when disrupted. Studying a patient’s own cells grown in the laboratory can further help researchers understand how changes in genes affect their function. Both animal and cellular models can be used to develop and test new drugs, which could provide treatment options for patients living with microphthalmia. This review summarises the key discoveries from animal and cellular models of microphthalmia and discusses how innovative new models can be used to further our understanding in the future.

Background and Purpose Voltage-gated sodium (Na-V) channels are expressed de novo in carcinomas where their activity promotes invasiveness. Breast and colon cancer cells express the neonatal splice variant of Na(V)1.5 (nNa(V)1.5), which has several amino acid substitutions in the domain I voltage-sensor compared with its adult counterpart (aNa(V)1.5). This study aimed to determine whether nNa(V)1.5 channels could be distinguished pharmacologically from aNa(V)1.5 channels. Experimental Approach Cells expressing either nNa(V)1.5 or aNa(V)1.5 channels were exposed to low MW inhibitors, an antibody or natural toxins, and changes in electrophysiological parameters were measured. Stable expression in EBNA cells and transient expression in Xenopus laevis oocytes were used. Currents were recorded by whole-cell patch clamp and two-electrode voltage-clamp, respectively. Key Results Several clinically used blockers of Na-V channels (lidocaine, procaine, phenytoin, mexiletine, ranolazine, and riluzole) could not distinguish between nNa(V)1.5 or aNa(V)1.5 channels. However, two tarantula toxins (HaTx and ProTx-II) and a polyclonal antibody (NESOpAb) preferentially inhibited currents elicited by either nNa(V)1.5 or aNa(V)1.5 channels by binding to the spliced region of the channel. Furthermore, the amino acid residue at position 211 (aspartate in aNa(V)1.5/lysine in nNa(V)1.5), that is, the charge reversal in the spliced region of the channel, played a key role in the selectivity, especially in antibody binding. Conclusion and Implications We conclude that the cancer-related nNa(V)1.5 channel can be distinguished pharmacologically from its nearest neighbour, aNa(V)1.5 channels. Thus, it may be possible to design low MW compounds as antimetastatic drugs for non-toxic therapy of nNa(V)1.5-expressing carcinomas.

Connexin-50 (Cx50) is among the most frequently mutated genes associated with congenital cataracts. While most of these disease-linked variants cause loss-of-function due to misfolding or aberrant trafficking, others directly alter channel properties. The mechanistic bases for such functional defects are mostly unknown. We investigated the functional and structural properties of a cataract-linked mutant, Cx50T39R (T39R), in the Xenopus oocyte system. T39R exhibited greatly enhanced hemichannel currents with altered voltage-gating properties compared to Cx50 and induced cell death. Co-expression of mutant T39R with wild-type Cx50 (to mimic the heterozygous state) resulted in hemichannel currents whose properties were indistinguishable from those induced by T39R alone, suggesting that the mutant had a dominant effect. Co-expression with Cx46 also produced channels with altered voltage-gating properties, particularly at negative potentials. All-atom molecular dynamics simulations indicate that the R39 substitution can form multiple electrostatic salt-bridge interactions between neighboring subunits that could stabilize the open-state conformation of the N-terminal domain, while also neutralizing the voltage-sensing residue D3 as well as residue E42 which participates in loop-gating. Together, these results suggest T39R acts as a dominant gain-of-function mutation that produces leaky hemichannels that may cause cytotoxicity in the lens and lead to development of cataracts.Statement of significanceWe investigated the functional and structural properties of a cataract-linked mutant, Cx50T39R (T39R), in the Xenopus oocyte system and showed that T39R exhibited greatly enhanced hemichannel currents with altered voltage-gating properties compared to Cx50 and induced cell death. Consistent with our experimental findings, all-atom equilibrium state molecular dynamics (MD) simulations of T39R show that R39 stabilized the open-state configuration of the N-terminal (NT) domain from an adjacent subunit. These results suggest that T39R causes disease by preventing the hemichannels from closing when present in the plasma membrane in the undocked state and provide an atomistic rationalization for the Cx50 disease-linked phenotype. They also expand our understanding of how connexin hemichannel channel gating is controlled.

In the embryonic heart development of mammals and birds, a straight initial heart tube undergoes left-handed helical looping, which is a remarkable and puzzling event. We are interested in the mechanism of this chiral helical looping. Recently, observations were reported that myocardial cells in the embryonic chick heart show intrinsic chirality of rotation. The chirality of myocardial cells, via anisotropic polarization of Golgi inside the cells, leads to a left-right (LR) asymmetry of cell shape. On cell boundaries of LR asymmetric cells, phosphorylated myosin and N-cadherin are enriched. Such LR asymmetric cellular circumstances lead to a large-scale three-dimensional chiral structure, the left-handed helical loop. However, the physical mechanism of this looping is unclear. Computer simulations were performed using a cell-based three-dimensional mathematical model assuming an anterior-rightward-biased contractile force of the cell boundaries on the ventral surface of the heart (orientation of a clock hand pointing to 10 to 11 o'clock). An initially straight heart tube was successfully remodeled to the left-handed helical tube via frequent convergent extension (CE) of collective cells, which corresponds to the previously reported observations of chick heart development. Although we assumed that the biased boundary contractile force was uniform all over the ventral side, orientations of the CEs became position specific on the anterior, posterior, right, and left regions on the ventral tube. Such position-specific CEs produced the left-handed helical loop. In addition, our results suggest the loop formation process consists of two distinct phases of preparation and explicit looping. Intrinsic cell properties of chirality in this investigation were discussed relating to extrinsic factors investigated by other researches. Finally, because CE is generally exerted in the axial developmental process across different animal species, we discussed the contribution of CE to the chiral heart structure across species of chick, mouse, Xenopus, and zebrafish.

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.

During nervous system development, axon bundles grow over large distances along well-defined pathways, guided by gradients of chemical cues in their environment. In addition, axons mechanically interact with their environment and respond to stiffness gradients they may encounter in the developing brain. In the model organism Xenopus laevis, retinal ganglion cell axons grow along the optic pathway toward the optic tectum. On their way through the mid-diencephalon, they turn caudally in response to gradients of the chemical guidance cues Semaphorin3A, Slit 1 and Slit 2 as well as tissue elasticity. However, while the importance of elasticity is becoming well-accepted in the context of development, biological tissues are not purely elastic but rather exhibit time-dependent mechanical properties known as viscoelasticity. In this project, I developed a method based on atomic force microscopy (AFM) to measure the viscoelasticity of biological tissues (in vivo Xenopus brain and ex vivo rodent spinal cord tissue). To investigate the influence of viscoelasticity on axon guidance, I first investigated the mechanical properties of the developing brain in vivo. I found that the Xenopus brain exhibited heterogeneous viscoelasticity and that greater tissue elasticity is correlated with a greater tissue viscosity. Subsequently, I wanted to understand how this heterogeneous tissue viscoelasticity may regulate axon growth. I developed tissue culture substrates with varying elasticities and viscosities and cultured Xenopus eye primordia explants on these substrates. I found that axons grew significantly longer on stiff elastic substrates compared to soft elastic substrates, reproducing previously published data, while there was no difference in axon length on soft and stiff viscoelastic substrates, indicating that viscosity is an important regulator of axon growth. I found that the regulation of axon length may be correlated with substrate timescale rather than by substrate elasticity. The substrate timescale is a measure of how long it takes a material to respond to an applied force or displacement; the shorter the timescale the faster the material response. Next, I used computer simulations to understand how the ratio of elastic and viscous tissue behaviour impacts axon growth. Exploiting the motor clutch model, which is well established for explaining the mechanical interactions of cells and their environment, I used physiological relevant mechanics I derived from the developing Xenopus brain and viscoelastic tissue culture substrates to simulate axon velocity, retrograde flow, and traction forces using elastic and viscoelastic frameworks. I found that the viscoelastic motor clutch model better described differences in axon lengths from the in vitro explant experiments. Furthermore, I found that the viscoelastic motor clutch model better describes axon velocities as a function of substrate timescale rather than the elastic framework. The characteristic substrate timescale appeared to be an important parameter in regulating axon growth. To further investigate if the viscoelastic motor clutch model was sufficient to explain axon growth, I investigated how traction forces and retrograde actin flow were regulated by substrate mechanics. To automate and simplify traction force microscopy (TFM) analysis, I developed custom software that enables users to perform TFM analysis with little prior understanding or knowledge. I found that growth cones mainly applied “pulling” forces to compliant substrates, and that the magnitude of the pulling force could be regulated by substrate timescale. Finally, I measured cytoskeletal dynamics of the growing axon and found that retrograde actin flow rates and microtubule polymerization rates were regulated by substrate mechanics. My results suggest that axon guidance is not only regulated by elasticity but also viscoelasticity and the viscous component should be considered when studying axon pathfinding. A better understanding of axon path finding could help us learn more about the role of mechanics in development and how to treat diseases in the central or peripheral nervous system.

It is known that in adult mammals, the heart has lost its regenerative capacity, making heart failure one of the leading causes of death worldwide. Previous research has demonstrated the regenerative ability of the heart of the adult Xenopus tropicalis, an anuran amphibian with a diploid genome and a close evolutionary relationship with mammals. Additionally, studies have shown that following ventricular apex resection, the heart can regenerate without scarring in X. tropicalis. Consequently, these previous results suggest that X. tropicalis is an appropriate alternative vertebrate model for the study of adult heart regeneration. A surgical model of cardiac regeneration in the adult X. tropicalis is presented herein. Briefly, the frogs were anesthetized and fixed; then, a small incision was made with iridectomy scissors, penetrating the skin and pericardium. Gentle pressure was applied to the ventricle, and the apex of the ventricle was then cut out with scissors. Cardiac injury and regeneration were confirmed by histology at 7-30 days post resection (dpr). This protocol established an apical resection model in adult X. tropicalis, which can be employed to elucidate the mechanisms of adult heart regeneration.

Amphibians have highly diverse sex-determining modes leading to a notable interest in vertebrate sex determination and sex chromosome evolution. The identification of sex-determining systems in amphibians, however, is often difficult as a vast majority consist of homomorphic sex chromosomes making them hard to distinguish. In this study, we used Diversity Array Technology sequencing (DArTseq) to identify the sex-determining system in the ornate burrowing frog from Australia, Platyplectrum ornatum. We applied DArTseq to 44 individuals, 19 males and 25 females, collected from two locations to develop sex-linked markers. Unexpectedly, these 44 individuals were classified into two distinct population clusters based on our SNP analyses, 36 individuals in cluster 1, and 8 individuals in cluster 2. We then performed sex-linkage analyses separately in each cluster. We identified 35 sex-linked markers from cluster 1, which were all associated with maleness. Therefore, P. ornatum cluster 1 is utilising a male heterogametic (XX/XY) sex-determining system. On the other hand, we identified 210 sex-linked markers from cluster 2, of which 89 were male specific, i.e., identifying XX/XY sex determining system and 111 were female specific, i.e., identifying ZZ/ZW sex determining system, suggesting existence of either male or female heterogametic sex determining system in cluster 2. We also performed cytogenetic analyses in 1 male and 1 female from cluster 1; however, we did not detect any visible differentiation between the X and Y sex chromosomes. We also mapped sex-linked markers from the two clusters against the P. ornatum genome and our comparative analysis indicated that the sex chromosomes in both clusters shared homologies to chromosome 10 (autosome) of Rana temporaria and ZWY sex chromosome of Xenopus tropicalis. Our preliminary data suggest that it is plausible that the cluster 2 has a potential to be either male or female heterogamety in sex determination, requiring further investigation.

The southern house mosquito, Culex quinquefasciatus, utilizes two odorant receptors, CquiOR10 and CquiOR2, narrowly tuned to oviposition attractants and well conserved among mosquito species. They detect skatole and indole, respectively, with reciprocal specificity. We swapped the transmembrane (TM) domains of CquiOR10 and CquiOR2 and identified TM2 as a specificity determinant. With additional mutations, we showed that CquiOR10A73L behaved like CquiOR2. Conversely, CquiOR2L74A recapitulated CquiOR10 specificity. Next, we generated structural models of CquiOR10 and CquiOR10A73L using RoseTTAFold and AlphaFold and docked skatole and indole using RosettaLigand. These modeling studies suggested space-filling constraints around A73. Consistent with this hypothesis, CquiOR10 mutants with a bulkier residue (Ile, Val) were insensitive to skatole and indole, whereas CquiOR10A73G retained the specificity to skatole and showed a more robust response than the wildtype receptor CquiOR10. On the other hand, Leu to Gly mutation of the indole receptor CquiOR2 reverted the specificity to skatole. Lastly, CquiOR10A73L, CquiOR2, and CquiOR2L74I were insensitive to 3-ethylindole, whereas CquiOR2L74A and CquiOR2L74G gained activity. Additionally, CquiOR10A73G gave more robust responses to 3-ethylindole than CquiOR10. Thus, we suggest the specificity of these receptors is mediated by a single amino acid substitution, leading to finely tuned volumetric space to accommodate specific oviposition attractants.

In the 1930s it was discovered that the Xenopus toad was a useful instrument in urine pregnancy tests. An injection of a woman’s urine into a toad that resulted in the production of ova was proof o...

Grain size is determined by the size and number of cells in the grain. The regulation of grain size is crucial for improving crop yield; however, the genes and molecular mechanisms that control grain size remain elusive. Here, we report that a member of the detoxification efflux carrier /Multidrug and Toxic Compound Extrusion (DTX/MATE) family transporters, BIG RICE GRAIN 1 (BIRG1), negatively influences grain size in rice (Oryza sativa L.). BIRG1 is highly expressed in reproductive organs and roots. In birg1 grain, the outer parenchyma layer cells of spikelet hulls are larger than in wild-type (WT) grains, but the cell number is unaltered. When expressed in Xenopus laevis oocytes, BIRG1 exhibits chloride efflux activity. Consistent with this role of BIRG1, the birg1 mutant shows reduced tolerance to salt stress at a toxic chloride level. Moreover, grains from birg1 plants contain a higher level of chloride than those of WT plants when grown under normal paddy field conditions, and the roots of birg1 accumulate more chloride than those of WT under saline conditions. Collectively, the data suggest that BIRG1 in rice functions as a chloride efflux transporter that is involved in mediating grain size and salt tolerance by controlling chloride homeostasis. This article is protected by copyright. All rights reserved.

Echinobase (www.echinobase.org) is a third generation web resource supporting genomic research on echinoderms. The new version was built by cloning the mature Xenopus model organism knowledgebase, Xenbase, refactoring data ingestion pipelines and modifying the user interface to adapt to multispecies echinoderm content. This approach leveraged over 15 years of previous database and web application development to generate a new fully featured informatics resource in a single year. In addition to the software stack, Echinobase uses the private cloud and physical hosts that support Xenbase. Echinobase currently supports six echinoderm species, focused on those used for genomics, developmental biology and gene regulatory network analyses. Over 38 000 gene pages, 18 000 publications, new improved genome assemblies, JBrowse genome browser and BLAST + services are available and supported by the development of a new echinoderm anatomical ontology, uniformly applied formal gene nomenclature, and consistent orthology predictions. A novel feature of Echinobase is integrating support for multiple, disparate species. New genomes from the diverse echinoderm phylum will be added and supported as data becomes available. The common code development design of the integrated knowledgebases ensures parallel improvements as each resource evolves. This approach is widely applicable for developing new model organism informatics resources.

In immature oocytes, Balbiani bodies are conserved membraneless condensates implicated in oocyte polarization, the organization of mitochondria, and long-term organelle and RNA storage. In Xenopus laevis, Balbiani body assembly is mediated by the protein Velo1. Velo1 contains an N-terminal prion-like domain (PLD) that is essential for Balbiani body formation. PLDs have emerged as a class of intrinsically disordered regions that can undergo various different types of intracellular phase transitions and are often associated with dynamic, liquid-like condensates. Intriguingly, the Velo1 PLD forms solid-like assemblies. Here we sought to understand why Velo1 phase behavior appears to be biophysically distinct from that of other PLD-containing proteins. Through bioinformatic analysis and coarse-grained simulations, we predict that the clustering of aromatic residues and the amino acid composition of residues between aromatics can influence condensate material properties, organization, and the driving forces for assembly. To test our predictions, we redesigned the Velo1 PLD to test the impact of targeted sequence changes in vivo. We found that the Velo1 design with evenly spaced aromatic residues shows rapid internal dynamics, as probed by fluorescent recovery after photobleaching, even when recruited into Balbiani bodies. Our results suggest that Velo1 might have been selected in evolution for distinctly clustered aromatic residues to maintain the structure of Balbiani bodies in long-lived oocytes. In general, our work identifies several tunable parameters that can be used to augment the condensate material state, offering a road map for the design of synthetic condensates.

Hyperpolarization-gated, cyclic nucleotide-activated (HCN1-4) channels are inwardly rectifying cation channels that display voltage dependent activation and de-activation. Pathogenic variants in HCN1 are associated with severe developmental and epileptic encephalopathies including the de novo HCN1 M305L variant. M305 is located in the S5 domain that is implicated in coupling voltage sensor domain movement to pore opening. This variant lacks voltage-dependent activation and de-activation and displays normal cation selectivity. To elucidate the impact of the mutation on the channel structure-function relations, molecular dynamics simulations of the wild type and mutant homotetramers were compared and identified a sulphur-aromatic interaction between M305 and F389 that contributes to the coupling of the voltage-sensing domain to the pore domain. To mimic the heterozygous condition as a heterotetrameric channel assembly, Xenopus oocytes were co-injected with various ratios of wild-type and mutant subunit cRNAs and the biophysical properties of channels with different subunit stoichiometries were determined. The results showed that a single mutated subunit was sufficient to significantly disrupt the voltage dependence of activation. The functional data were qualitatively consistent with predictions of a model that assumes independent activation of the voltage sensing domains allosterically controlling the closed to open transition of the pore. Overall, the M305L mutation results in an HCN1 channel that lacks voltage dependence and facilitates excitatory cation flow at membrane potentials that would normally close the channel. Our findings provide molecular insights into HCN1 channels and reveal the structural and biophysical basis of the severe epilepsy phenotype associated with the M305L mutation.

The Notch pathway is an ancient intercellular signaling system with crucial roles in numerous cell-fate decision processes across species. While the canonical pathway is activated by ligand-induced cleavage and nuclear localization of membrane-bound Notch, Notch can also exert its activity in a ligand/transcription-independent fashion, which is conserved in Drosophila, Xenopus, and mammals. However, the noncanonical role remains poorly understood in in vivo processes. Here we show that increased levels of the Notch intracellular domain (NICD) in the early mesoderm inhibit heart development, potentially through impaired induction of the second heart field (SHF), independently of the transcriptional effector RBP-J. Similarly, inhibiting Notch cleavage, shown to increase noncanonical Notch activity, suppressed SHF induction in embryonic stem cell (ESC)-derived mesodermal cells. In contrast, NICD overexpression in late cardiac progenitor cells lacking RBP-J resulted in an increase in heart size. Our study suggests that noncanonical Notch signaling has stage-specific roles during cardiac development.

Messenger RNA (mRNA) can be transported and targeted to different subcellular compartments and locally translated. Local translation is an evolutionally conserved mechanism that in mammals, provides an important tool to exquisitely regulate the subcellular proteome in different cell types, including neurons. Local translation in axons is involved in processes such as neuronal development, function, plasticity, and diseases. Here, we summarize the current progress on axonal mRNA transport and translation. We focus on the regulatory mechanisms governing how mRNAs are transported to axons and how they are locally translated in axons. We discuss the roles of axonally synthesized proteins, which either function locally in axons, or are retrogradely trafficked back to soma to achieve neuron-wide gene regulation. We also examine local translation in neurological diseases. Finally, we give a critical perspective on the remaining questions that could be answered to uncover the fundamental rules governing local translation, and discuss how this could lead to new therapeutic targets for neurological diseases.

Neuroligin (NLG) protein is a nerve cell adhesion molecule and plays a key role in the precision apposition of presynaptic domains on inhibitory and excitatory synapses. Existing studies mainly focused on the function of NLG3 against the excitatory channel. However, the interaction between insect NLG3 and ionotropic GABA receptor, which is the main inhibitory channel, remains unclear. In this study, the Nlg3 of common cutworm (CCW), Spodoptera litura Fabricius, one important agricultural Lepidopteron, is selected to explore its function in the inhibitory channel.The SlNlg3 was obtained and the SlNLG3 contains the characteristic features including transmembrane domain, PDZ-binding motif and type-B carboxylesterases signature 2 motif. The SlNlg3 messenger RNA (mRNA) was most abundant in midgut, and exhibited multiple expression patterns in different developmental stages and tissues or body parts. Compared with the single injection of SlRDL1, the median effective concentration value of GABA in activating currents was smaller in Xenopus laevis oocytes co-injected with SlRDL1 and SlNlg3. In addition, SlNlg3 could enhance the GABA-induced current of homomeric SlRDL1 channel from -391.86 ± 15.41 to -2152.51 ± 30.09 nA. DsSlNlg3 depressed the expression level of SlNlg3 mRNA more than 64.29% at 6 h. After exposure to median lethal dose of fluralaner, the mortality of CCW injected with dsSlNlg3 was significantly decreased by 13.34% and 30.00% at 24 and 48 h, respectively, compared to injection of dsEGFP.NLG3 should have physiological function on ionotropic GABA receptor in vitro, which provided a favorable foundation for further research on the physiological function of Nlg gene in Lepidopteron. © 2021 Society of Chemical Industry.

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.

Crypsis increases survival by reducing predator detection. Xenopus laevis tadpoles decode light properties from the substrate to induce two responses: a cryptic coloration response where dorsal skin pigmentation is adjusted to the colour of the substrate (background adaptation) and a behavioural crypsis where organisms move to align with a specific colour surface (background preference). Both processes require organisms to detect reflected light from the substrate. We explored the relationship between background adaptation and preference and the light properties able to trigger both responses. We also analysed which retinal photosensor (type II opsin) is involved. Our results showed that these two processes are segregated mechanistically, as there is no correlation between the preference for a specific background with the level of skin pigmentation, and different dorsal retina-localized type II opsins appear to underlie the two crypsis modes. Indeed, inhibition of melanopsin affects background adaptation but not background preference. Instead, we propose pinopsin is the photosensor involved in background preference. pinopsin mRNA is co-expressed with mRNA for the sws1 cone photopigment in dorsally located photoreceptors. Importantly, the developmental onset of pinopsin expression aligns with the emergence of the preference for a white background, but after the background adaptation phenotype appears. Furthermore, white background preference of tadpoles is associated with increased pinopsin expression, a feature that is lost in premetamorphic froglets along with a preference for a white background. Thus, our data show a mechanistic dissociation between background adaptation and background preference, and we suggest melanopsin and pinopsin, respectively, initiate the two responses.

Sperm matrix metalloproteinase-2 (MMP-2) is necessary for frog fertilization. Monospermy is ensured by a fast, electrical block to polyspermy mediated by a positive fertilization potential. To determine the role of the MMP-2 hemopexin domain (HPX) in a fast block to polyspermy during fertilization of the frog, Xenopus tropicalis, we prepared mutant frogs deficient in mmp2 gene using the transcription activator-like effector nuclease method. mmp2 ΔHPX (-/-) sperm without MMP-2 protein were able to fertilize wild-type (WT; +/+) eggs. However, polyspermy occurred in some eggs. The mutant sperm generated a normal fertilization potential amounting to 10 mV, and were able to fertilize eggs at 10 mV, at which WT sperm never fertilized. Sensitivity during voltage-dependent fertilization decreased in mutant sperm. This study demonstrates for the first time that the genetic alteration of the MMP-2 molecule in sperm causes polyspermy during fertilization of a monospermic species. Our findings provide reliable evidence that sperm MMP-2 is indispensable for the fast, electrical block to polyspermy during Xenopus fertilization.

Microplastics and associated adverse effects have been on the global agenda in recent years. Because of its importance as a model organism for studies on developmental biology, Xenopus laevis has been chosen as the study animal in in vitro teratogenesis studies. FETAX test uses early-stage embryos of X. laevis to measure the potential of substances to cause mortality, malformation, and growth inhibition in developing embryos. The aim of this study was to examine the effects of high molecular weight polyvinyl chloride (HMW-PVC) on parental X. laevis frogs and their embryos using the FETAX test. To this purpose, a HMW-PVC dose of 1% of body weight/twice each week was provided to frogs by oral gavage throughout 6 weeks. After the procedure, oocytes and sperms of HMW-PVC-exposed frogs were fertilized and FETAX was applied to selected embryos. After the completion of a 96-h incubation period, tadpoles were examined, their live/dead status were determined, their lengths were measured, and their anomalies were photographed. Besides, excised organs of the parental frogs were referred to histopathology examination. On the other hand, the mRNA expression levels of Hsp70, Myf5, Bmp4, Pax6, and Esr1 genes were determined by applying real-time quantitative PCR method to cDNA which was synthesized from the total RNA of embryos. The results showed that treatment with HMW-PVC dose of 1% of body weight/twice each week caused malformations and decreased viability. Hsp70 and Pax6 gene expression levels significantly decreased in all assay groups, as compared with controls. Lung and intestine tissues showed normal appearance in histopatological examination. Further research is required to explain the whole effects of HMW-PVC exposure on X. laevis embryos.

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.

Summary Stomatal closure is an important process to prevent water loss in plants response to drought stress, which is finely modulated by ion channels together with their regulators in guard cells, especially the S‐type anion channel AtSLAC1 in Arabidopsis. However, the functional characterization and regulation analyses of anion channels in gramineous crops, such as in maize guard cells are still limited. In this study, we identified an S‐type anion channel ZmSLAC1 that was preferentially expressed in maize guard cells and involved in stomatal closure under drought stress. We found that two Ca2+‐dependent protein kinases ZmCPK35 and ZmCPK37 were expressed in maize guard cells and localized on the plasma membrane. Lesion of ZmCPK37 resulted in drought‐sensitive phenotypes. Mutation of ZmSLAC1 and ZmCPK37 impaired ABA‐activated S‐type anion currents in maize guard cells, while the S‐type anion currents were increased in the guard cells of ZmCPK35‐ and ZmCPK37‐overexpression lines. Electrophysiological characterization in maize guard cells and Xenopus oocytes indicated that ZmCPK35 and ZmCPK37 could activate ZmSLAC1‐mediated Cl‐ and NO3 ‐ currents. The maize inbred and hybrid lines overexpressing ZmCPK35 and ZmCPK37 exhibited enhanced tolerance and increased yield under drought conditions. In conclusion, our results demonstrate that ZmSLAC1 plays crucial roles in stomatal closure in maize, whose activity is regulated by ZmCPK35 and ZmCPK37. Elevation of ZmCPK35 and ZmCPK37 expression levels is a feasible way to improve maize drought tolerance as well as reduce yield loss under drought stress.

The poly-Immunoglobulin Receptor (pIgR) transcytoses J chain-containing antibodies through mucosal epithelia. In mammals, two cis-duplicates of PIGR, FCMR and FCAMR, flank the PIGR gene. A PIGR duplication is first found in amphibians, previously annotated as PIGR2 (herein xlFCAMR), and is expressed by antigen-presenting cells. We demonstrate that xlFcamR is the equivalent of mammalian FcamR. It has been assumed that pIgR is the oldest member of this family, yet our data could not distinguish whether PIGR or FCAMR emerged first; however, FCMR was the last family member to emerge. Interestingly, bony fish 'pIgR' is not an orthologue of tetrapod pIgR, and possibly acquired its function via convergent evolution. PIGR/FCAMR/FCMR are members of a larger superfamily including TREM, CD300, and NKp44, which we name the 'double-disulfide Ig superfamily' (ddIgSF). Domains related to each ddIgSF family were identified in cartilaginous fish (sharks, chimeras) and encoded in a single gene cluster syntenic to the human pIgR locus. Thus, the ddIgSF families date back to earliest antibody-based adaptive immunity, but apparently not before. Finally, our data strongly suggest that the J chain arose in evolution only for Ig multimerization. This study provides a framework for further studies of pIgR and the ddIgSF in vertebrates. This article is protected by copyright. All rights reserved.

Vertebrate hearts have undergone marked morphological and structural changes to adapt to different environments and lifestyles as part of the evolutionary process. Amphibians were the first vertebrates to migrate to land. Transition from aquatic to terrestrial environments required the ability to circulate blood against the force of gravity. In this study, we investigated the passive mechanical properties and histology of the ventricles of three species of Anura (frogs and toads) from different habitats, Xenopus laevis (aquatic), Pelophylax nigromaculatus (semiaquatic), and Bufo japonicus formosus (terrestrial). Pressure-loading tests demonstrated stiffer ventricles of P. nigromaculatus and B. j. formosus compared X. laevis ventricles. Histological analysis revealed a remarkable difference in the structure of cardiac tissue: thickening of the compact myocardium layer of P. nigromaculatus and B. j. formosus and enrichment of the collagen fibers of B. j. formosus. The amount of collagen fibers differed among the species, as quantitatively confirmed by second-harmonic generation light microscopy. No significant difference was observed in cardiomyocytes isolated from each animal, and the sarcomere length was almost the same. The results indicate that the ventricles of Anura stiffen during adaptation to life on land. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10867-021-09579-4.

Gene encoding aspartyl dipeptidase from Xenopus levies (PepExl) is upregulated by thyroid hormone and is proposed to play a significant role in resorption of tadpole tail during metamorphosis. However, the importance of peptidase activity for the resorption of the tail remain elusive. Here we report the crystal structures of first eukaryotic S51 peptidase, PepExl, in its ligand-free and Asp-bound states at 1.4 and 1.8 Å resolutions, respectively. The active site is located at dimeric interface and the catalytic triad is found to be dissembled in ligand-free and assembled in Asp-bound state. Structural comparison and molecular dynamic simulations of ligand-free and Asp-bound states shows that distinct loop (loop-A) plays an important role in active site shielding, substrate binding and enzyme activation. This study illuminates the Asp-X dipeptide binding in PepExl is associated with ordering of the loop-A and assembly of residues of catalytic triad in active conformation for enzymatic activity.

During mitosis, spindle assembly relies on centrosomal and acentrosomal microtubule nucleation pathways that all require the γ-Tubulin Ring Complex (γ-TuRC) and its adaptor protein NEDD1. The activity of these different pathways needs to be coordinated to ensure bipolar spindle assembly ( Cavazza et al., 2016) but the underlying mechanism is still unclear. Previous studies have identified three sites in NEDD1 (S377, S405 and S411) that when phosphorylated drive MT nucleation at the centrosomes, around the chromosomes and on pre-existing MTs respectively ( Lüders et al., 2006; Pinyol et al., 2013; Sdelci et al., 2012). Here we aimed at getting additional insights into the mechanism that coordinates the different MT nucleation pathways in dividing cells using a collection of HeLa stable inducible cell lines expressing NEDD1 phospho-variants at these three sites and Xenopus egg extracts. Our results provide further support for the essential role of phosphorylation at the three residues. Moreover, we directly demonstrate that S411 phosphorylation is essential for MT branching using TIRF microscopy in Xenopus egg extracts and we show that it plays a crucial role in ensuring the balance between centrosome and chromosome-dependent MT nucleation required for bipolar spindle assembly in mitotic cells.

Corticosteroids are so vital for organ maturation that reduced corticosteroid signaling during postembryonic development causes death in terrestrial vertebrates. Indeed, death occurs at metamorphosis in frogs lacking proopiomelanocortin (pomc) or the glucocorticoid receptor (GR; nr3c1). Some residual corticosteroids exist in pomc mutants to activate the wild-type (WT) GR and mineralocorticoid receptor (MR), and the elevated corticosteroids in GR mutants may activate MR. Thus, we expected a more severe developmental phenotype in tadpoles with inactivation of 21-hydroxylase, which should eliminate all interrenal corticosteroid biosynthesis. Using CRISPR/Cas9 in Xenopus tropicalis, we produced an 11-base pair deletion in cyp21a2, the gene encoding 21-hydroxylase. Growth and development were delayed in cyp21a2 mutant tadpoles, but unlike the other frog models, they survived metamorphosis. Consistent with an absence of 21-hydroxylase, mutant tadpoles had a 95% reduction of aldosterone in tail tissue, but they retained some corticosterone (∼40% of WT siblings), an amount, however, too low for survival in pomc mutants. Decreased corticosteroid signaling was evidenced by reduced expression of corticosteroid-response gene, klf9, and by impaired negative feedback in the hypothalamus-pituitary-interrenal axis with higher messenger RNA expression levels of crh, pomc, star, and cyp11b2 and an approximately 30-fold increase in tail content of progesterone. In vitro tail-tip culture showed that progesterone can transactivate the frog GR. The inadequate activation of GR by corticosterone in cyp21a2 mutants was likely compensated for by sufficient corticosteroid signaling from other GR ligands to allow survival through the developmental transition from aquatic to terrestrial life.

Thyroid hormones (TH) and glucocorticoids (GC) are involved in numerous developmental and physiological processes. The effects of individual hormones are well documented, but little is known about the joint actions of the two hormones. To decipher the crosstalk between these two hormonal pathways, we conducted a transcriptional analysis of genes regulated by TH, GC, or both hormones together in liver of

The Tet-ON system is an important molecular tool for temporally and spatially-controlled inducible gene expression. Here, we developed a Tet-ON system to induce transgene expression specifically in the rod photoreceptors of medaka fish. Our modified reverse tetracycline-controlled transcriptional transactivator (rtTAm) with 5 amino acid substitutions dramatically improved the leakiness of the transgene in medaka fish. We generated a transgenic line carrying a self-reporting vector with the rtTAm gene driven by the Xenopus rhodopsin promoter and a tetracycline response element (TRE) followed by the green fluorescent protein (GFP) gene. We demonstrated that GFP fluorescence was restricted to the rod photoreceptors in the presence of doxycycline in larval fish (9 days post-fertilization). The GFP fluorescence intensity was enhanced with longer durations of doxycycline treatment up to 72 h and in a dose-dependent manner (5-45 μg/ml). These findings demonstrate that the Tet-ON system using rtTAm allows for spatiotemporal control of transgene expression, at least in the rod photoreceptors, in medaka fish.

Ladybird homeobox (Lbx) transcription factors have crucial functions in muscle and nervous system development in many animals. Amniotes have two Lbx genes, but only Lbx1 is expressed in spinal cord. In contrast, teleosts have three lbx genes and we show here that zebrafish lbx1a, lbx1b and lbx2 are expressed by distinct spinal cell types, and that lbx1a is expressed in dI4, dI5 and dI6 interneurons, as in amniotes. Our data examining lbx expression in Scyliorhinus canicula and Xenopus tropicalis suggest that the spinal interneuron expression of zebrafish lbx1a is ancestral, whereas lbx1b has acquired a new expression pattern in spinal cord progenitor cells. lbx2 spinal expression was probably acquired in the ray-finned lineage, as this gene is not expressed in the spinal cords of either amniotes or S. canicula. We also show that the spinal function of zebrafish lbx1a is conserved with mouse Lbx1. In zebrafish lbx1a mutants, there is a reduction in the number of inhibitory spinal interneurons and an increase in the number of excitatory spinal interneurons, similar to mouse Lbx1 mutants. Interestingly, the number of inhibitory spinal interneurons is also reduced in lbx1b mutants, although in this case the number of excitatory interneurons is not increased. lbx1a;lbx1b double mutants have a similar spinal interneuron phenotype to lbx1a single mutants. Taken together these data suggest that lbx1b and lbx1a may be required in succession for correct specification of dI4 and dI6 spinal interneurons, although only lbx1a is required for suppression of excitatory fates in these cells.Research Highlightslbx1 spinal expression and function is conserved in vertebrates. In contrast, zebrafish lbx1b and lbx2 have novel spinal expression patterns that probably evolved in the ray-finned vertebrate lineage (lbx2) or teleosts (lbx1b).

Amphibians such as Xenopus tropicalis exhibit a remarkable capacity for tissue regeneration after traumatic injury. Although transforming growth factor-β (TGF-β) receptor signaling is known to be essential for tissue regeneration in fish and amphibians, the role of TGF-β ligands in this process is not well understood. Here, we show that inhibition of TGF-β1 function prevents tail regeneration in Xenopus tropicalis tadpoles. We found that expression of tgfb1 is present before tail amputation and is sustained throughout the regeneration process. CRISPR-mediated knock-out (KO) of tgfb1 retards tail regeneration; the phenotype of tgfb1 KO tadpoles can be rescued by injection of tgfb1 mRNA. Cell proliferation, a critical event for the success of tissue regeneration, is downregulated in tgfb1 KO tadpoles. In addition, tgfb1 KO reduces the expression of phosphorylated Smad2/3 (pSmad2/3) which is important for TGF-β signal-mediated cell proliferation. Collectively, our results show that TGF-β1 regulates cell proliferation through the activation of Smad2/3. We therefore propose that TGF-β1 plays a critical role in TGF-β receptor-dependent tadpole tail regeneration in Xenopus.

Creatine (Cr)/phosphocreatine has the ability to buffer the high-energy phosphate, thereby contributing to intracellular energy homeostasis. As Cr biosynthetic enzyme deficiency is reported to increase susceptibility to colitis under conditions of inflammatory stress, Cr is critical for maintaining intestinal homeostasis under inflammatory stress. Cr is mainly produced in the hepatocytes and then distributed to other organs of the body by the circulatory system. Since monocarboxylate transporter 9 (MCT9) and monocarboxylate transporter 12 (MCT12) have been reported to accept Cr as a substrate, these transporters are proposed as candidates for Cr efflux transporter in the liver. The aim of this study was to elucidate the transport mechanism on Cr supply from the hepatocytes. Immunohistochemical staining of the rat liver sections revealed that both MCT9 and MCT12 were localized on the sinusoidal membrane of the hepatocytes. In the transport studies using Xenopus laevis oocyte expression system, [14C]Cr efflux from MCT9- or MCT12-expressing oocytes was significantly greater than that from water-injected oocytes. [14C]Cr efflux from primary cultured hepatocytes was significantly decreased following MCT12 mRNA knockdown, whereas this efflux was not decreased after mRNA knockdown of MCT9. Based on the extent of MCT12 protein downregulation and Cr efflux after knockdown of MCT12 in primary cultured rat hepatocytes, the contribution ratio of MCT12 in Cr efflux was calculated as 76.4%. Our study suggests that MCT12 substantially contributes to the efflux of Cr at the sinusoidal membrane of the hepatocytes.NEW & NOTEWORTHY Our study is the first to identify the role of monocarboxylate transporter 12 (MCT12) as a transporter of creatine (Cr) in the liver. MCT12 was found to significantly contribute to the efflux of Cr on the sinusoidal membrane of the hepatocytes. Since hepatocytes are known to be involved in creatine biosynthesis, the present findings can be beneficial for the regulation of Cr biosynthesis and supply.

Characeae are closely related to the ancient algal ancestors of all land plants. The long characean cells display a pH banding pattern to facilitate inorganic carbon import in the acid zones for photosynthetic efficiency. The excess OH−, generated in the cytoplasm after CO2 is taken into the chloroplasts, is disposed of in the alkaline band. To identify the transporter responsible, we searched the Chara australis transcriptome for homologues of mouse Slc4a11, which functions as OH−/H+ transporter. We found a single Slc4-like sequence CL5060.2 (named CaSLOT). When CaSLOT was expressed in Xenopus oocytes, an increase in membrane conductance and hyperpolarization of resting potential difference (PD) was observed with external pH increase to 9.5. These features recall the behavior of Slc4a11 in oocytes and are consistent with the action of a pH-dependent OH−/H+ conductance. The large scatter in the data might reflect intrinsic variability of CaSLOT transporter activation, inefficient expression in the oocyte due to evolutionary distance between ancient algae and frogs, or absence of putative activating factor present in Chara cytoplasm. CaSLOT homologues were found in chlorophyte and charophyte algae, but surprisingly not in related charophytes Zygnematophyceae or Coleochaetophyceae.

Cells are remarkably resilient structures, they are able to recover from injuries to their plasma membrane (PM) and cytoskeleton that would normally constitute existential threats. This capacity is exemplified by Xenopus laevis oocytes which can recover from very large PM defects through exocytotic and endocytic events and can repair damaged cortical cytoskeleton structures through the formation of a contractile actomyosin ring (AMR). Formation of the AMR involves the localized Ca2+ -dependent activation of RhoA and Cdc42, and the pre-patterning of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). However, this model fails to account for observations that suggest a link between cytoskeletal dynamics, intracellular tension and AMR formation. It also does not explain why the formation of an AMR is not involved in the cytoskeletal repair program of adherent cells. We show here evidence for the support of tension as an essential regulatory signal for the formation of AMR. Indeed, oocytes in which global tension has been experimentally reduced were unable to form a functional AMR following injury, showing severely diminished RhoA activity at the wound site. These new insights place the cytoskeleton at the center of events involving changes in cell shape such as cytokinesis which also involves the formation and closure of an AMR. This article is protected by copyright. All rights reserved.

The ER or donut-like structures localized aquaporin NIP5;1, which interacts with PIPs and alters their localization from plasma membrane to donut-like structures, regulates water permeability. NOD26-like intrinsic proteins (NIPs) play important roles in nutrient uptake and response to various stresses. However, there have been few studies of their functions in water transportation in citrus. Here, we demonstrate the functions of a novel citrus NIP aquaporin (CsNIP5;1) via multiple physiological and biochemical experiments. CsNIP5;1 showed high water permeability when expressed in Xenopus laevis oocytes and yeast. However, subcellular localization assays showed that this protein was localized in the endoplasmic reticulum (ER) or donut-like structures in citrus callus and tobacco leaf. Meanwhile, overexpression of CsNIP5;1 led to a reduction in the water permeability of citrus callus. Protein-protein interaction experiments and subcellular localization assays further revealed that CsNIP5;1 physically interacted with PIPs (CsPIP1;1 and AtPIP2;1), which altered their subcellular localization from the plasma membrane to donut-like structures. Together, CsNIP5;1 was identified as a good water channel when expressed in oocytes and yeast. Meanwhile, CsNIP5;1 participated in the regulation of water permeability of citrus callus, which may be associated with CsNIP5;1-induced re-localization of water channels PIPs. In summary, these results provide new insights into the regulatory mechanism of AQPs-mediated water diffusion.

α-Conotoxins are small disulfide-rich peptides found in the venom of marine cone snails and are potent antagonists of nicotinic acetylcholine receptors (nAChRs). They are valuable pharmacological tools and have potential therapeutic applications for the treatment of chronic pain or neurological diseases and disorders. In the present study, we synthesized and functionally characterized a novel α-conotoxin Bt1.8, which was cloned from Conus betulinus. Bt1.8 selectively inhibited ACh-evoked currents in Xenopus oocytes expressing rat(r) α6/α3β2β3 and rα3β2 nAChRs with an IC50 of 2.1 nM and 9.4 nM, respectively, and similar potency for human (h) α6/α3β2β3 and hα3β2 nAChRs. Additionally, Bt1.8 had higher binding affinity with a slower dissociation rate for the rα6/α3β2β3 subtype compared to rα3β2. The amino acid sequence of Bt1.8 is significantly different from other reported α-conotoxins targeting the two nAChR subtypes. Further Alanine scanning analyses demonstrated that residues Ile9, Leu10, Asn11, Asn12 and Asn14 are critical for its inhibitory activity at the α6/α3β2β3 and α3β2 subtypes. Moreover, the NMR structure of Bt1.8 indicated the presence of a relatively larger hydrophobic zone than other α4/7-conotoxins which may explain its potent inhibition at α6/α3β2β3 nAChRs.

Mammalian oocytes undergo major changes in zinc content and localization in order to be fertilized, the most striking being the rapid exocytosis of over ten billion zinc ions, known as zinc sparks. Here we report that fertilization of amphibian Xenopus laevis eggs also initiates a zinc spark that progresses across the cell surface in coordination with dynamic calcium waves. This zinc exocytosis is accompanied by a newly recognized loss of intracellular manganese: synchrotron-based X-ray fluorescence and analytical electron microscopy reveal that zinc and manganese are sequestered in a system of cortical granules that are abundant at the animal pole. Through Electron-Nuclear Double-Resonance (ENDOR) studies, we rule out Mn2+ complexation with phosphate or nitrogenous ligands in intact eggs but the data are consistent with a carboxylate coordination environment. Our observations suggest that zinc and manganese fluxes are a conserved feature of fertilization in vertebrates and that they function as part of a physiological block to polyspermy., Graphical abstract