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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]

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]

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]

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]

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]

Xenopus is one of the essential model systems for studying vertebrate development. However, one drawback of this system is that, because of the opacity of Xenopus embryos, 3D imaging analysis is limited to surface structures, explant cultures, and post‐embryonic tadpoles. To develop a technique for 3D tissue/organ imaging in whole Xenopus embryos, we identified optimal conditions for using placental alkaline phosphatase (PLAP) as a transgenic reporter and applied it to the correlative light microscopy and block‐face imaging (CoMBI) method for visualization of PLAP‐expressing tissues/organs. In embryos whose endogenous alkaline phosphatase activities were heat‐inactivated, PLAP staining visualized various tissue‐specific enhancer/promoter activities in a manner consistent with green fluorescent protein (GFP) fluorescence. Furthermore, PLAP staining appeared to be more sensitive than GFP fluorescence as a reporter, and the resulting expression patterns were not mosaic, in striking contrast to the mosaic staining pattern of β‐galactosidase expressed from the lacZ gene that was introduced by the same transgenesis method. Owing to efficient penetration of alkaline phosphatase substrates, PLAP activity was detected in deep tissues, such as the developing brain, spinal cord, heart, and somites, by whole‐mount staining. The stained embryos were analyzed by the CoMBI method, resulting in the digital reconstruction of 3D images of the PLAP‐expressing tissues. These results demonstrate the efficacy of the PLAP reporter system for detecting enhancer/promoter activities driving deep tissue expression and its combination with the CoMBI method as a powerful approach for 3D digital imaging analysis of specific tissue/organ structures in Xenopus embryos. [ABSTRACT FROM AUTHOR]

Wnt is a family of secreted signaling proteins involved in the regulation of cellular processes, including maintenance of stem cells, carcinogenesis, and cell differentiation. In the context of early vertebrate embryogenesis, graded distribution of Wnt proteins has been thought to regulate positional information along the antero‐posterior axis. However, understanding of the molecular basis for Wnt spatial distribution remains poor. Modified states of heparan sulfate (HS) proteoglycans are essential for Wnt8 localization, because depletion of N‐deacetylase/N‐sulfotransferase 1 (NDST1), a modification enzyme of HS chains, decreases Wnt8 levels and NDST1 overexpression increases Wnt8 levels on the cell surface. Since overexpression of NDST1 increases both deacetylation and N‐sulfation of HS chains, it is not clear which function of NDST1 is actually involved in Wnt8 localization. In the present study, we generated an NDST1 mutant that specifically increases deacetylation, but not N‐sulfation, of HS chains in Xenopus embryos. Unlike wild‐type NDST1, this mutant did not increase Wnt8 accumulation on the cell surface, but it reduced canonical Wnt signaling, as determined with the TOP‐Flash reporter assay. These results suggest that N‐sulfation of HS chains is responsible for localization of Wnt8 and Wnt8 signaling, whereas deacetylation has an inhibitory effect on canonical Wnt signaling. Consistently, overexpression of wild‐type NDST1, but not the mutant, resulted in small eyes in Xenopus embryos. Thus, our NDST1 mutant enables us to dissect the regulation of Wnt8 localization and signaling by HS proteoglycans by specifically manipulating the enzymatic activities of NDST1. [ABSTRACT FROM AUTHOR]

The dorsoventral gradient of BMP signaling plays an essential role in embryonic patterning. Zinc Finger SWIM-Type Containing 4 (zswim4) is expressed in the Spemann-Mangold organizer at the onset of Xenopus gastrulation and is then enriched in the developing neuroectoderm at the mid-gastrula stages. Knockdown or knockout of zswim4 causes ventralization. Overexpression of zswim4 decreases, whereas knockdown of zswim4 increases the expression levels of ventrolateral mesoderm marker genes. Mechanistically, ZSWIM4 attenuates the BMP signal by reducing the protein stability of SMAD1 in the nucleus. Stable isotope labeling by amino acids in cell culture (SILAC) identifies Elongin B (ELOB) and Elongin C (ELOC) as the interaction partners of ZSWIM4. Accordingly, ZSWIM4 forms a complex with the Cul2-RING ubiquitin ligase and ELOB and ELOC, promoting the ubiquitination and degradation of SMAD1 in the nucleus. Our study identifies a novel mechanism that restricts BMP signaling in the nucleus. Synopsis: Zswim4 fine-tunes BMP signaling in Xenopus early embryonic patterning by interacting with the Cul2-RING ubiquitin ligase, Elongin B and C, thereby forming a protein disruption complex that ultimately reduces nuclear Smad1 protein stability. Zswim4 is a novel inhibitor of the BMP signaling pathway, localized in the nucleus. Zswim4 physically interacts with Smad1 through the Smad1 MH1 domain. Zswim4 reduces Smad1 stability and increases Smad1 ubiquitination in the nucleus. Zswim4 interacts with Cullin2 and Elongin B and C, forming a protein disruption complex to promote Smad1 ubiquitination. Zswim4 fine-tunes BMP signaling in Xenopus early embryonic patterning by interacting with the Cul2-RING ubiquitin ligase, Elongin B and C, thereby forming a protein disruption complex that ultimately reduces nuclear Smad1 protein stability. [ABSTRACT FROM AUTHOR]

The African clawed frog, Xenopus laevis, and the Western clawed frog, Xenopus tropicalis, have been foundational model organisms for establishing key principles of embryonic development. Today, the utility of Xenopus has been greatly expanded for studying a wide range of biological processes both in health and disease. Here, we describe the latest advancements from the Xenopus community, which span the molecular, cellular, tissue, and organismal scales. Key Findings: Instrumental in elucidating the mechanisms of early vertebrate development and functional genomics of birth defects. Central to understanding the cellular and molecular basis of regeneration. Key model to study cell, cilia, and mechano‐biology. [ABSTRACT FROM AUTHOR]

Modeling metastasis in animal systems has been an important focus for developing cancer therapeutics. Xenopus laevis is a well‐established model, known for its use in identifying genetic mechanisms underlying diseases and disorders in humans. Prior literature has suggested that the drug, ivermectin, can be used in Xenopus to induce melanocytes to convert into a metastatic melanoma‐like state, and thus could be ideal for testing possible melanoma therapies in vivo. However, there are notable inconsistencies between ivermectin studies in Xenopus and the application of ivermectin in mammalian systems, that are relevant to cancer and melanoma research. In this review, we examine the ivermectin‐induced phenotypes in Xenopus, and we explore the current uses of ivermectin in human research. We conclude that while ivermectin may be a useful drug for many biomedical purposes, it is not ideal to induce a metastatic melanocyte phenotype in Xenopus for testing the effects of potential melanoma therapeutics. [ABSTRACT FROM AUTHOR]

Amphibians generally have three types of pigment cells, namely, melanophores (black and brown), xanthophores (yellow and red), and iridophores (iridescent). Single knockout of the tyr, slc2a7, and hps6 genes in Xenopus tropicalis results in the absence of melanophores, xanthophores, and iridophores, respectively. The generation of triple‐ knockout (3KO) X. tropicalis for these three genes could allow for observation of internal organs without sacrificing the animals, which would be transparent due to the absence of pigments. In this study, we generated 3KO X. tropicalis, which is one of the most widely used model amphibians, through crossing of a slc2a7 single‐knockout frog with a tyr and hps6 double‐knockout frog, followed by intercrossing of their offspring. The 3KO tadpoles had transparent bodies like the nop mutant and the frogs had translucent bodies. This translucency allowed us to observe the heart, lungs, stomach, liver, and digestive tract through the ventral body skin without surgery. After intravital staining, 3KO X. tropicalis showed much clearer fluorescent signals of mineralized tissues compared with the wild type. These 3KO X. tropicalis provide a useful mutant line for continuous observation of internal organs and fluorescent signals in the body. In particular, such 3KO frogs would revolutionize fluorescence monitoring in transgenic tadpoles and frogs expressing fluorescent proteins. [ABSTRACT FROM AUTHOR]

Background: Members of the sulfotransferase superfamily (SULT) influence the activity of a wide range of hormones, neurotransmitters, metabolites and xenobiotics. However, their roles in developmental processes are not well characterized even though they are expressed during embryogenesis. We previously found in a microarray screen that Six1 up‐regulates LOC100037047, which encodes XB5850668.L, an uncharacterized sulfotransferase. Results: Since Six1 is required for patterning the embryonic ectoderm into its neural plate, neural crest, preplacodal and epidermal domains, we used loss‐ and gain‐of function assays to characterize the role of XB5850668.L during this process. Knockdown of endogenous XB5850668.L resulted in the reduction of epidermal, neural crest, cranial placode and otic vesicle gene expression domains, concomitant with neural plate expansion. Increased levels had minimal effects, but infrequently expanded neural plate and neural crest gene domains, and infrequently reduced cranial placode and otic vesicle gene domains. Mutation of two key amino acids in the sulfotransferase catalytic domain required for PAPS binding and enzymatic activity tended to reduce the effects of overexpressing the wild‐type protein. Conclusions: Our analyses indicates that XB5850668.L is a member of the SULT2 family that plays important roles in patterning the embryonic ectoderm. Some aspects of its influence likely depend on sulfotransferase activity. Key Findings: XB5850668.L is an uncharacterized sulfotransferase It is a member of the SULT2 family It is required for proportioning the embryonic ectoderm Some of its effects require sulfotransferase activity. [ABSTRACT FROM AUTHOR]

Cover Photograph: Overexpression of wild type NDST1 accumulates mV‐Wnt8 on cell surfaces (white margin between NDST1 expressing (red) and non‐expressing cells (black) of Xenopus embryo (see Suzuki et al. pp 248‐255). [ABSTRACT FROM AUTHOR]

Since CRISPR‐based genome editing technology works effectively in the diploid frog Xenopus tropicalis, a growing number of studies have successfully modeled human genetic diseases in this species. However, most of their targets were limited to non‐syndromic diseases that exhibit abnormalities in a small fraction of tissues or organs in the body. This is likely because of the complexity of interpreting the phenotypic variations resulting from somatic mosaic mutations generated in the founder animals (crispants). In this study, we attempted to model the syndromic disease campomelic dysplasia (CD) by generating sox9 crispants in X. tropicalis. The resulting crispants failed to form neural crest cells at neurula stages and exhibited various combinations of jaw, gill, ear, heart, and gut defects at tadpole stages, recapitulating part of the syndromic phenotype of CD patients. Genotyping of the crispants with a variety of allelic series of mutations suggested that the heart and gut defects depend primarily on frame‐shift mutations expected to be null, whereas the jaw, gill, and ear defects could be induced not only by such mutations but also by in‐frame deletion mutations expected to delete part of the jawed vertebrate‐specific domain from the encoded Sox9 protein. These results demonstrate that Xenopus crispants are useful for investigating the phenotype–genotype relationships behind syndromic diseases and examining the tissue‐specific role of each functional domain within a single protein, providing novel insights into vertebrate jaw evolution. [ABSTRACT FROM AUTHOR]

Aquaporins are important transmembrane water transport proteins which transport water and several neutral molecules. However, how aquaporins are involved in the synergistic transport of Mg2+ and water remains poorly understood. Here, we found that the cassava aquaporin MePIP2;7 was involved in Mg2+ transport through interaction with MeMGT9, a lower affinity magnesium transporter protein. Knockdown of MePIP2;7 in cassava led to magnesium deficiency in basal mature leaves with chlorosis and necrotic spots on their edges and starch over‐accumulation. Mg2+ content was significantly decreased in leaves and roots of MePIP2;7‐RNA interference (PIP‐Ri) plants grown in both field and Mg2+‐free hydroponic solution. Xenopus oocyte injection analysis verified that MePIP2;7 possessed the ability to transport water only and MeMGT9 was responsible for Mg2+ efflux. More importantly, MePIP2;7 improved the transportability of Mg2+ via MeMGT9 as verified using the CM66 mutant complementation assay and Xenopus oocytes expressing system. Yeast two‐hybrid, bimolecular fluorescence complementation, co‐localization, and co‐immunoprecipitation assays demonstrated the direct protein–protein interaction between MePIP2;7 and MeMGT9 in vivo. Mg2+ flux was significantly elevated in MePIP2;7‐overexpressing lines in hydroponic solution through non‐invasive micro‐test technique analysis. Under Mg2+‐free condition, the retarded growth of PIP‐Ri transgenic plants could be recovered with Mg2+ supplementation. Taken together, our results demonstrated the synergistic effect of the MePIP2;7 and MeMGT9 interaction in regulating water and Mg2+ absorption and transport in cassava. [ABSTRACT FROM AUTHOR]

CMG (Cdc45‐MCM‐GINS) helicase assembly at the replication origin is the culmination of eukaryotic DNA replication initiation. This process can be reconstructed in vitro using defined factors in Saccharomyces cerevisiae; however, in vertebrates, origin‐dependent CMG formation has not yet been achieved partly due to the lack of a complete set of known initiator proteins. Since a microcephaly gene product, DONSON, was reported to remodel the CMG helicase under replication stress, we analyzed its role in DNA replication using a Xenopus cell‐free system. We found that DONSON was essential for the replisome assembly. In vertebrates, DONSON physically interacted with GINS and Polε via its conserved N‐terminal PGY and NPF motifs, and the DONSON‐GINS interaction contributed to the replisome assembly. DONSON's chromatin association during replication initiation required the pre‐replicative complex, TopBP1, and kinase activities of S‐CDK and DDK. Both S‐CDK and DDK required DONSON to trigger replication initiation. Moreover, human DONSON could substitute for the Xenopus protein in a cell‐free system. These findings indicate that vertebrate DONSON is a novel initiator protein essential for CMG helicase assembly. Synopsis: Vertebrate DONSON is involved in replication fork stability as a component of the replisome complex. This study reveals that DONSON also functions as an initiator protein to promote CMG (Cdc45‐MCM‐GINS) helicase assembly through the interaction with GINS in the initiation of DNA replication. DONSON is required for CMG helicase assembly during DNA replication initiation.DONSON physically interacts with Cdc45, GINS, and Polε via its N‐terminal disordered region.The DONSON‐GINS interaction contributes to CMG helicase assembly.DONSON's chromatin association during replication initiation requires the pre‐replicative complex, TopBP1, and kinase activities of S‐CDK and DDK.S‐CDK and DDK require DONSON function to trigger replication initiation. [ABSTRACT FROM AUTHOR]

The author comments on a study that demonstrates how a mutation in the anion chloride channel ClC-K-barttin affects an endocytic YxxØ motif and uses expression in Xenopus oocytes and mammalian cells to link increased stability of the ClC-K-barttin complex. He discusses the finding that the barttin point mutation left protein expression levels and subcellular distribution unaffected under control conditions. He explains the significance of linking protein functions to organ physiology.

Janine Ziermann-Canabarro is an established researcher in the field of biology, specifically in the areas of development and evolution, head muscles, and the heart. She has received prestigious awards for her papers on cranial muscle development and muscle identity in evolutionary morphology. Ziermann-Canabarro studied biology at Friedrich Schiller University in Jena, Germany, under the guidance of Prof. Dr. Lennart Olsson. Her interest in biology stemmed from her love for animals and the interconnectedness of life through evolution. She credits Dr. Julia C. Boughner as her most influential mentor, who has guided her in research, leadership, and outreach. Ziermann-Canabarro's favorite paper is on avian craniofacial muscles, which inspired her to pursue cranial muscle research. She believes that the future of evolutionary developmental biology (EvoDevo) lies in studying zoonoses and their impact on development and evolution, as well as utilizing new methodologies and datasets. Her advice to junior EvoDevo researchers is to not hesitate in asking questions and seeking opportunities. [Extracted from the article]

The vertebrate organizer is a specified embryonic tissue that regulates dorsoventral patterning and axis formation. Although numerous cellular signaling pathways have been identified as regulators of the organizer's dynamic functions, the process remains incompletely understood, and as‐yet unknown pathways remain to be explored for sophisticated mechanistic understanding of the vertebrate organizer. To identify new potential key factors of the organizer, we performed complementary DNA (cDNA) microarray screening using organizer‐mimicking Xenopus laevis tissue. This analysis yielded a list of prospective organizer genes, and we determined the role of six‐transmembrane domain containing transmembrane protein 150b (Tmem150b) in organizer function. Tmem150b was expressed in the organizer region and induced by Activin/Nodal signaling. In X. laevis, Tmem150b knockdown resulted in head defects and a shortened body axis. Moreover, Tmem150b negatively regulated bone morphogenetic protein (BMP) signaling, likely via physical interaction with activin receptor‐like kinase 2 (ALK2). These findings demonstrated that Tmem150b functions as a novel membrane regulatory factor of BMP signaling with antagonistic effects, contributing to the understanding of regulatory molecular mechanisms of organizer axis function. Investigation of additional candidate genes identified in the cDNA microarray analysis could further delineate the genetic networks of the organizer during vertebrate embryogenesis. Significance Statement: During vertebrate embryogenesis, the Spearman organizer regulates germ layer specification and embryonic axis patterning. Identifying the underlying molecular mechanisms in the organizer is essential to understand the sophisticated process of embryogenesis. Still, there are numerous unexplored genes expressed at the organizer which can be important to describe better molecular machineries during development or related biological contexts. To discover novel genes expressed at the organizer, we performed microarray by using the organizer‐mimicking Xenopus tissues. In this study, we found potential organizer genes and we further characterized tmem150b from the list. Finally, we showed Tmem150b is expressed at the organizer and it is a novel antagonizing factor of bone morphogenetic protein signaling during Xenopus development. [ABSTRACT FROM AUTHOR]

Xenopus tadpoles serve as an exceptional model organism for studying post‐embryonic development in vertebrates. During post‐embryonic development, large‐scale changes in tissue morphology, including organ regeneration and metamorphosis, occur at the organ level. However, understanding these processes in a three‐dimensional manner remains challenging. In this study, the use of X‐ray micro‐computed tomography (microCT) for the three‐dimensional observation of the soft tissues of Xenopus tadpoles was explored. The findings revealed that major organs, such as the brain, heart, and kidneys, could be visualized with high contrast by phosphotungstic acid staining following fixation with Bouin's solution. Then, the changes in brain shape during telencephalon regeneration were analyzed as the first example of utilizing microCT to study organ regeneration in Xenopus tadpoles, and it was found that the size of the amputated telencephalon recovered to >80% of its original length within approximately 1 week. It was also observed that the ventricles tended to shrink after amputation and maintained this state for at least 3 days. This shrinkage was transient, as the ventricles expanded to exceed their original size within the following week. Temporary shrinkage and expansion of the ventricles, which were also observed in transgenic or fluorescent dye‐injected tadpoles with telencephalon amputation, may be significant in tissue homeostasis in response to massive brain injury and subsequent repair and regeneration. This established method will improve experimental analyses in developmental biology and medical science using Xenopus tadpoles. [ABSTRACT FROM AUTHOR]

Over the previous decade, numerous new approach methodologies (NAMs) have been developed and validated for the detection of endocrine activity of individual chemicals or environmental samples. These NAMs can be largely separated into three categories, in silico tools, in vitro assays, and in vivo assays using organisms or life stages not considered as laboratory animals, each with their own advantages and disadvantages. While in vitro assays provide more mechanistic information, the use of whole organisms such as fish or amphibian embryos provides a more holistic view of the net effects of an environmental sample on hormonal activity. A panel of bioassays was used to test the endocrine activity of several samples from the Danube River at Novi Sad, Serbia. The results of the in vitro assays have been published previously. Here, we present the results of the in vivo assays that were performed at the same time on the same samples. These whole organism assays are based on the use of transgenic fish and amphibian eleuthero‐embryos and included the Xenopus Eleuthero‐embryo Thyroid Assay (XETA), the Rapid Estrogen ACTivity In Vivo assay (REACTIV), and the Rapid Androgen Disruption Activity Reporter (RADAR) assay. Discrepancies between the different in vitro assays have previously been reported. The results of the in vivo studies also indicate discrepancies between the in vivo and in vitro data with an underestimation of the endocrine activity by the in vitro tests. Therefore, a battery of tests is advised with the initial diagnostic performed with in vivo tests to cover a wider range of modes of action and to allow the appropriate in vitro assay(s) to be selected to confirm the mode of action. Practitioner Points: Endocrine activity was quantified in surface water using in vitro and in vivo models.The in vivo results fit with previously reported in vitro results.Higher activity was observed in water samples with in vivo models, which cover a wider range of modes of action.Endocrine activity of surface water samples may be underestimated when measured with in vitro models. [ABSTRACT FROM AUTHOR]

New approach methods (NAMs) are increasingly important to help accelerate the pace of ecological risk assessment and offer more ethical, affordable, and efficient alternatives to traditional toxicity tests. In the present study, we describe the development, technical characterization, and initial testing of a toxicogenomics tool, EcoToxChip (384‐well quantitative polymerase chain reaction [qPCR] array), to support chemical management and environmental monitoring for three laboratory model species—fathead minnow (Pimephales promelas), African clawed frog (Xenopus laevis), and Japanese quail (Coturnix japonica). Chip design, including gene selection, was informed by a diverse end‐user group and quality control metrics (e.g., primer assay, reverse transcription, and PCR efficiency) performed well based on a priori established criteria. Correlation with RNA sequencing (seq) data provided additional confidence in this novel toxicogenomics tool. Although the present study represents an initial testing of only 24 EcoToxChips for each of the model species, the results provide increased confidence in the robustness/reproducibility of EcoToxChips for evaluating perturbations in gene expression associated with chemical exposure and thus, this NAM, combined with early‐life stage toxicity testing, could augment current efforts for chemical prioritization and environmental management. Environ Toxicol Chem 2023;42:1763–1771. © 2023 SETAC [ABSTRACT FROM AUTHOR]

Summary: Transport of K+ to the xylem is a key process in the mineral nutrition of the shoots. Although CIPK‐CBL complexes have been widely shown to regulate K+ uptake transport systems, no information is available about the xylem ones. Here, we studied the physiological roles of the voltage‐gated K+ channel SlSKOR and its regulation by the SlCIPK23‐SlCBL1/9 complexes in tomato plants.We phenotyped gene‐edited slskor and slcipk23 tomato knockout mutants and carried out two‐electrode voltage‐clamp (TEVC) and BiFC assays in Xenopus oocytes as key approaches.SlSKOR was preferentially expressed in the root stele and was important not only for K+ transport to shoots but also, indirectly, for that of Ca2+, Mg2+, Na+, NO3−, and Cl−. Surprisingly, the SlCIPK23‐SlCBL1/9 complexes turned out to be negative regulators of SlSKOR. Inhibition of SlSKOR by SlCIPK23‐SlCBL1/9 was observed in Xenopus oocytes and tomato plants. Regulation of SKOR‐like channels by CIPK23‐CBL1 complexes was also present in Medicago, grapevine, and lettuce but not in Arabidopsis and saltwater cress.Our results provide a molecular framework for coordinating root K+ uptake and its translocation to the shoot by SlCIPK23‐SlCBL1/9 in tomato plants. Moreover, they evidenced that CIPK‐CBL‐target networks have evolved differently in land plants. [ABSTRACT FROM AUTHOR]

The normal stages of embryonic development for wild‐type Xenopus laevis were established by Nieuwkoop and Faber in 1956, a milestone in the history of understanding embryonic development. However, this work lacked photographic images and staining for skeleton structures from the corresponding stages. Here, we provide high‐quality images of embryonic morphology and skeleton development to facilitate studies on amphibian development. On the basis of the classical work, we selected the albino mutant of X. laevis as the observation material to restudy embryonic development in this species. The lower level of pigmentation makes it easier to interpret histochemical experiments. At 23°C, albino embryos develop at the same rate as wild‐type embryos, which can be divided into 66 stages as they develop into adults in about 58 days. We described the complete embryonic development system for X. laevis, supplemented with pictures of limb and skeleton development that are missing from previous studies, and summarized the characteristics and laws of limb and skeleton development. Our study should aid research into the development of X. laevis and the evolution of amphibians. [ABSTRACT FROM AUTHOR]

Somatic and germline gain‐of‐function point mutations in RAF, one of the first oncogenes to be discovered in humans, delineate a group of tumor‐prone syndromes known as the RASopathies. In this study, we document the first human phenotype resulting from the germline loss‐of‐function of the proto‐oncogene RAF1 (a.k.a. CRAF). In a consanguineous family, we uncovered a homozygous p.Thr543Met variant segregating with a neonatal lethal syndrome with cutaneous, craniofacial, cardiac, and limb anomalies. Structure‐based prediction and functional tests using human knock‐in cells showed that threonine 543 is essential to: (i) ensure RAF1's stability and phosphorylation, (ii) maintain its kinase activity toward substrates of the MAPK pathway, and (iii) protect from stress‐induced apoptosis mediated by ASK1. In Xenopus embryos, mutant RAF1T543M failed to phenocopy the effects of normal and overactive FGF/MAPK signaling, confirming its hypomorphic activity. Collectively, our data disclose the genetic and molecular etiology of a novel lethal syndrome with progeroid features, highlighting the importance of RTK signaling for human development and homeostasis. Synopsis: A novel recessive variant in RAF1 was identified as a possible cause of a hitherto unknown lethal neonatal syndrome with progeroid features. This private p.Thr543Met mutation abolished downstream ERK pathway signaling while increasing susceptibility to apoptosis via ASK1 de‐repression. A homozygous p.Thr543Met mutation in RAF1 was found in a neonate presenting with a syndrome encompassing cutaneous, craniofacial, cardiac and limb anomalies.In cultured cells and in vivo using Xenopus assays the p.Thr543Met mutation behaved as a loss‐of‐function allele that abolishes downstream ERK pathway signaling in response to growth factor stimulation.p.Thr543Met knock‐in in 293T cells revealed that RAF1T543M is unstable and more sensitive to stress‐induced apoptosis via ASK1 de‐repression.These findings describe, for the first time, the consequences of a loss of RAF1 function during human development, contrasting with gain‐of‐function mutations seen in cancer‐prone RASopathies. [ABSTRACT FROM AUTHOR]

Cryptochromes (CRYs) are multifunctional molecules that act as a circadian clock oscillating factor, a blue‐light sensor, and a light‐driven magnetoreceptor. Cry genes are classified into several groups based on the evolutionary relationships. Cryptochrome 6 gene (Cry6) is present in invertebrates and lower vertebrates such as amphibians and fishes. Here we identified a Cry6 ortholog in Xenopus tropicalis (XtCry6). XtCRY6 retains a conserved long N‐terminal extension (termed CRY N‐terminal extension; CNE) that is not found in any CRY in the other groups. A structural prediction suggested that CNE contained unique structures; a tetrahelical fold structure topologically related to KaiA/RbsU domain, overlapping nuclear‐ and nucleolar‐localizing signals (NLS/NoLS), and a novel motif (termed DI‐UIM) overlapping a double‐sided ubiquitin‐interacting motif (DUIM) and an inverted ubiquitin‐interacting motif (IUIM). Potential activities of the NLS/NoLS and DI‐UIM were examined to infer the molecular function of XtCRY6. GFP‐NLS/NoLS fusion protein exogenously expressed in HEK293 cells was mostly observed in the nucleolus, while GFP‐XtCRY6 was observed in the cytoplasm. A glutathione S‐transferase (GST) pull‐down assay suggested that the DI‐UIM physically interacts with polyubiquitin. Consistently, protein docking simulations implied that XtCRY6 DI‐UIM binds two ubiquitin molecules in a relationship of a twofold rotational symmetry with the symmetry axis parallel or perpendicular to the DI‐UIM helix. These results strongly suggested that XtCRY6 does not function as a circadian transcriptional repressor and that it might have another function such as photoreceptive molecule regulating light‐dependent protein degradation or gene expression through a CNE‐mediated interaction with ubiquitinated proteins in the cytoplasm and/or nucleolus. [ABSTRACT FROM AUTHOR]

Ultraviolet B (UVB) in sunlight cause skin damage, ranging from wrinkles to photoaging and skin cancer. UVB can affect genomic DNA by creating cyclobutane pyrimidine dimers (CPDs) and pyrimidine–pyrimidine (6–4) photoproducts (6–4PPs). These lesions are mainly repaired by the nucleotide excision repair (NER) system and by photolyase enzymes that are activated by blue light. Our main goal was to validate the use of Xenopus laevis as an in vivo model system for investigating the impact of UVB on skin physiology. The mRNA expression levels of xpc and six other genes of the NER system and CPD/6–4PP photolyases were found at all stages of embryonic development and in all adult tissues tested. When examining Xenopus embryos at different time points after UVB irradiation, we observed a gradual decrease in CPD levels and an increased number of apoptotic cells, together with an epidermal thickening and an increased dendricity of melanocytes. We observed a quick removal of CPDs when embryos are exposed to blue light versus in the dark, confirming the efficient activation of photolyases. A decrease in the number of apoptotic cells and an accelerated return to normal proliferation rate was noted in blue light‐exposed embryos compared with their control counterparts. Overall, a gradual decrease in CPD levels, detection of apoptotic cells, thickening of epidermis, and increased dendricity of melanocytes, emulate human skin responses to UVB and support Xenopus as an appropriate and alternative model for such studies. [ABSTRACT FROM AUTHOR]

We characterized Xenopus laevis C‐C motif chemokine ligand 19.L (ccl19.L) and C‐C motif chemokine ligand 21.L (ccl21.L) during early Xenopus embryogenesis. The temporal and spatial expression patterns of ccl19.L and ccl21.L tended to show an inverse correlation, except that the expression level was higher in the dorsal side at the gastrula stage. For example, even at the dorsal sector of the gastrulae, ccl19.L was expressed in the axial region and ccl21.L was expressed in the paraxial region. Dorsal overexpression of ccl19.L and ccl21.L and knockdown of Ccl19.L and Ccl21.L inhibited gastrulation, but their functions were different in cell behaviors during morphogenesis. Observation of Keller sandwich explants revealed that overexpression of both ccl19.L and ccl21.L and knockdown of Ccl21.L inhibited the convergent extension movements, while knockdown of Ccl19.L did not. ccl19.L‐overexpressing explants attracted cells at a distance and ccl21.L‐overexpressing explants attracted neighboring cells. Ventral overexpression of ccl19.L and ccl21.L induced secondary axis‐like structures and chrd.1 expression at the ventral side. Upregulation of chrd.1 was induced by ligand mRNAs through ccr7.S. Knockdown of Ccl19.L and Ccl21.L inhibited gastrulation and downregulated chrd.1 expression at the dorsal side. The collective findings indicate that ccl19.L and ccl21.L might play important roles in morphogenesis and dorsal–ventral patterning during early embryogenesis in Xenopus. [ABSTRACT FROM AUTHOR]

Neural tissue is derived from three precursor regions: neural plate, neural crest, and preplacodal ectoderm. These regions are determined by morphogen‐mediated signaling. Morphogen distribution is generally regulated by binding to an extracellular matrix component, heparan sulfate (HS) proteoglycan. HS is modified by many enzymes, such as N‐deacetyl sulfotransferase 1 (Ndst1), which is highly expressed in early development. However, functions of HS modifications in ectodermal patterning are largely unknown. In this study, we analyzed the role of Ndst1 using Xenopus embryos. We found that ndst1 was expressed in anterior neural plate and the trigeminal region at the neurula stage. ndst1 overexpression expanded the neural crest (NC) region, whereas translational inhibition reduced not only the trigeminal region, but also the adjacent NC region, especially the anterior part. At a later stage, ndst1 knocked‐down embryos showed defects in cranial ganglion formation. We also found that Ndst1 activates Wnt signaling pathway at the neurula stage. Taken together, our results suggest that N‐sulfonated HS accumulates Wnt ligand and activates Wnt signaling in ndst1‐expressing cells, but that it inhibits signaling in non‐ndst1‐expressing cells, leading to proper neuroectodermal patterning. [ABSTRACT FROM AUTHOR]

Marine teleosts ingest large amounts of seawater containing various ions, including 0.4 mM boric acid, which can accumulate at toxic levels in the body. However, the molecular mechanisms by which marine teleosts absorb and excrete boric acid are not well understood. Aquaporins (Aqps) are homologous to the nodulin‐like intrinsic protein (NIP) family of plant boric acid channels. To investigate the potential roles of Aqps on boric acid transport across the plasma membrane in marine teleosts, we analyzed the function of Aqps of Japanese pufferfish (Takifugu rubripes) expressed in Xenopus laevis oocytes. Takifugu genome database contains 16 genes encoding the aquaporin family members (aqp0a, aqp0b, aqp1aa, aqp1ab, aqp3a, aqp4a, aqp7, aqp8bb, aqp9a, aqp9b, aqp10aa, aqp10bb, aqp11a, aqp11b, aqp12, and aqp14). When T. rubripes Aqps (TrAqps) were expressed in X. laevis oocytes, a swelling assay showed that boric acid permeability was significantly increased in oocytes expressing TrAqp3a, 7, 8bb, 9a, and 9b. The influx of boric acid into these oocytes was also confirmed by elemental quantification. Electrophysiological analysis using a pH microelectrode showed that these TrAqps increase B(OH)3 permeability. These results indicate that TrAqp3a, 7, 8bb, 9a, and 9b act as boric acid transport systems, likely as channels, in marine teleosts. [ABSTRACT FROM AUTHOR]

Background: Xenopus frogs are used extensively for modeling genetic diseases owing to characteristics such as the abundance of eggs combined with their large size, allowing easy manipulation, and rapid external embryo development enabling the examination of cellular and phenotypic alterations throughout embryogenesis. However, genotyping of mutant animals is currently done either as part of a large group, requiring many embryos, or late in development with welfare effects. Therefore, we adapted the Zebrafish Embryonic Genotyper for rapid genomic DNA extraction from Xenopus tropicalis and Xenopus laevis at early stages. Results: Sufficient and good quality DNA was extracted as early as the Nieuwkoop and Faber stage 19 and, importantly, no detrimental effects of the extraction process on the subsequent tadpole development, behavior, or morphology were observed. Amplicons of up to 800 bp were successfully amplified and used for further analyses such as gel electrophoresis, T7 endonuclease I assay and Sanger sequencing. Conclusion: This method allows rapid genotyping during the early stages of Xenopus development, which enables safe identification of mutants. These can be analyzed at early developmental stages or selected for raising without the need for invasive genotyping later, with resource savings and ethical gains in line with the 3Rs principles. Key Findings: Xenopus frogs are used extensively for modelling genetic diseases. However, current procedures for genotyping of mutant animals require many embryos, or are done late in development, which hampers experiments have welfare effects. Therefore, we developed a method allowing rapid genotyping during the early stages of Xenopus development, which has no detrimental effects on the subsequent tadpole growth, with resource savings and ethical gains in line with the 3Rs principles. [ABSTRACT FROM AUTHOR]

The primary objective of the present study was to examine the influence of early systemic toxicity resulting from copper (Cu) exposure on metamorphic processes in Xenopus laevis. A 28‐day exposure study with copper, initiated at developmental stage 10, was performed using test concentrations of 3.0, 9.0, 27.2, 82.5, and 250 μg Cu/L. The primary endpoints included mortality, developmental stage, embryo‐larval malformation, behavioral effects, hindlimb length (HLL), growth (snout‐vent length [SVL] and wet body weight), and histopathology. The 28‐day LC50 value with 95% confidence intervals was 61.2 (51.4–72.9) μg Cu/L with 250 μg Cu/L resulting in complete lethality. Developmental arrest in the 82.5 and delay in the 27.2 μg Cu/L treatments was observed as early as study day 10 continuing throughout the remainder of exposure. SVL‐normalized HLL, body weight, and SVL in the 27.2 and 82.5 μg Cu/L treatments were significantly decreased relative to control. At 82.5 μg Cu/L, and thyroid gland size was markedly reduced when compared with controls consistent with the stage of developmental and growth arrest. Concentration‐dependent findings in the intestine, liver, gills, eyes, and pharyngeal mucosa were consistent with non‐endocrine systemic toxicity. These were prevalent in the 9.0 and 27.2 μg Cu/L treatment groups but were minimally evident or absent in the 82.5 μg/L group, which was attributed to developmental arrest. In conclusion, developmental delay in larvae exposed to 27.2 and 82.5 μg Cu/L was the result of systemic toxicity occurring in early development prior hypothalomo‐pituitary‐thyroid axis (HPT)‐driven metamorphosis and was not indicative of endocrine disruption. The primary objective of the present study was to examine the influence of early systemic toxicity resulting from copper (Cu) exposure on metamorphic processes in Xenopus laevis. A 28‐day exposure study with copper, initiated at developmental stage 10, was performed using test concentrations of 3.0, 9.0, 27.2, 82.5, and 250 μg Cu/L. Developmental delay in larvae exposed to 27.2 and 82.5 μg Cu/L was the result of systemic toxicity occurring in early development prior hypothalomo‐pituitary‐thyroid axis (HPT)‐driven metamorphosis and was not indicative of endocrine disruption. [ABSTRACT FROM AUTHOR]

The oxygen environment in African clawed frogs (Xenopus laevis) continuously changes during their development, which involves a rapid increase in the body size, metamorphosis, and transition to adulthood. Nevertheless, there are limited reports on experimental models that are available for studying fluctuations in the oxygen environment in X. laevis. Thus, this study aimed to develop an experimental model on intermittent hypoxia in X. laevis and evaluate hypoxia and oxidative stress in the same. X. laevis were submerged in water with a dissolved oxygen concentration of 2 mg/L for 30 min; they were then removed from the water and allowed to freely absorb oxygen for 5 min. Immunostaining of pimonidazole‐containing frozen tissue sections of the lung and liver using anti‐pimonidazole antibodies as the hypoxia probes revealed that more than 95% of the submerged X. laevis cells were pimonidazole positive, providing direct evidence of tissue hypoxia. When the amount of oxidative stress in the lungs and liver was evaluated in terms of the amount of lipid peroxides, the diving group showed a 2.08‐fold and 3.20‐fold increase over the normal group, respectively. Following hypoxia exposure, the dry‐to‐wet weight ratios of the lung tissues was 1.27 times higher (p <.05), while the liver tissues was 1.06 times higher (although not significant). Thus, the degree of damage depended on the tissues affected. In the future, we believe that this model will be a promising option for analyzing the physiological responses of X. laevis to hypoxia and oxidative stress. [ABSTRACT FROM AUTHOR]

The tetraspanins (Tspans) constitute a family of cell surface proteins with four transmembrane domains. Tspans have been found on the plasma membrane and on exosomes of various organelles. Reports on the function of Tspans during the early development of Xenopus have mainly focused on the expression of uroplakins in gametes. Although the roles of extracellular vesicles (EVs) including exosomes have been actively analyzed in cancer research, the contribution of EVs to early development is not well understood. This is because the diffusivity of EVs is not compatible with a very strict developmental process. In this study, we analyzed members of the Tspan family in early development of Xenopus. Expression was prominent in specific organs such as the notochord, eye, cranial neural crest cells (CNCs), trunk neural crest cells, placodes, and somites. We overexpressed several combinations of Tspans in CNCs in vitro and in vivo. Changing the partner changed the distribution of fluorescent‐labeled Tspans. Therefore, it is suggested that expression of multiple Tspans in a particular tissue might produce heterogeneity of intercellular communication, which has not yet been recognized. [ABSTRACT FROM AUTHOR]

Background: The thyroid hormones—thyroxine (T4) and 3,5,3′triiodothyronine (T3)—regulate the development of the central nervous system (CNS) in vertebrates by acting in different cell types. Although several T3 target genes have been identified in the brain, the changes in the transcriptome in response to T3 specifically in neural stem and progenitor cells (NSPCs) during the early steps of NSPCs activation and neurogenesis have not been studied in vivo. Here, we characterized the transcriptome of FACS‐sorted NSPCs in response to T3 during Xenopus laevis metamorphosis. Results: We identified 1252 upregulated and 726 downregulated genes after 16 hours of T3 exposure. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that T3‐upregulated genes were significantly enriched in rRNA processing and maturation, protein folding, ribosome biogenesis, translation, mitochondrial function, and proteasome. These results suggest that NSPCs activation induced by T3 is characterized by an early proteome remodeling through the synthesis of the translation machinery and the degradation of proteins by the proteasome. Conclusion: This work provides new insights into the dynamics of activation of NPSCs in vivo in response to T3 during a critical period of neurogenesis in the metamorphosis. Key Findings: We characterized the transcriptome of Xenopus neural stem and progenitor cells (NSPCs) in response to 16 hours of T3 exposure.T3‐upregulated genes were significantly enriched in ribosome biogenesis, proteasome and mitochondrial function.Our results reveal new insights into the early dynamics of NSPCs activation during neurogenesis. [ABSTRACT FROM AUTHOR]

Canonical Wnt signalling plays important roles in early embryogenesis, such as axis formation due to its activation and head formation due to its inhibition. ß‐catenin protein stability is a key factor in canonical Wnt signalling. Several E3 ubiquitin ligases contribute to ß‐catenin degradation through the ubiquitin/proteasome system. We characterised an E3 ubiquitin ligase gene, Xenopus laevis macrophage erythroblast attacher (maea), during early development. maea transcripts were ubiquitously detected in early embryos. The expression levels of the Wnt target genes nodal homolog 3, gene 1 (nodal3.1), and siamois homeodomain 1 (sia1), which were induced by injection with ß‐catenin mRNA, were reduced by maea.S mRNA co‐injection. maea.S overexpression at the anterior dorsal region enlarged head structures, whereas Maea knockdown interfered with head formation in Xenopus embryos. Maea.S decreased and ubiquitinated ß‐catenin protein. ß‐catenin‐4KRs protein, which mutated the four lysine (K) residues known as ubiquitinated sites to arginine (R) residues, was also ubiquitinated and degraded by Maea.S. These findings suggest that Maea contributes to β‐catenin degradation by ubiquitination of unknown lysine residues in early Xenopus development. [ABSTRACT FROM AUTHOR]

Thyroid hormone (T3) is essential for normal development and metabolism, especially during postembryonic development, a period around birth in mammals when plasma T3 levels reach their peak. T3 functions through two T3 receptors, TRα and TRβ. However, little is known about the tissue‐specific functions of TRs during postembryonic development because of maternal influence and difficulty in manipulation of mammalian models. We have studied Xenopus tropicalis metamorphosis as a model for human postembryonic development. By using TRα knockout (Xtr·thratmshi) tadpoles, we have previously shown that TRα is important for T3‐dependent intestinal remodeling and hindlimb development but not tail resorption during metamorphosis. Here, we have identified genes bound by TR in premetamorphic wild‐type and Xtr·thratmshi tails with or without T3 treatment by using chromatin immunoprecipitation–sequencing and compared them with those in the intestine and hindlimb. Compared to other organs, the tail has much fewer genes bound by TR or affected by TRα knockout. Bioinformatic analyses revealed that among the genes bound by TR in wild‐type but not Xtr·thratmshi organs, fewer gene ontology (GO) terms or biological pathways related to metamorphosis were enriched in the tail compared to those in the intestine and hindlimb. This difference likely underlies the drastic effects of TRα knockout on the metamorphosis of the intestine and hindlimb but not the tail. Thus, TRα has tissue‐specific roles in regulating T3‐dependent anuran metamorphosis by directly targeting the pathways and GO terms important for metamorphosis. [ABSTRACT FROM AUTHOR]

Size of the nucleus, a membrane‐bound organelle for DNA replication and transcription in eukaryotic cells, varies to adapt nuclear functions to the surrounding environment. Nuclear size strongly correlates with cytoplasmic size and genomic content. Previous studies using Xenopus laevis have unraveled two modes, cytoplasmic and chromatin‐based mechanisms, for controlling nuclear size. However, owing to limited comparative analyses of the mechanisms among eukaryotic species, the contribution of each mechanism in controlling nuclear size has not been comprehensively elucidated. Here, we compared the relative contribution utilizing a cell‐free reconstruction system from the cytoplasmic extract of unfertilized eggs of Xenopus tropicalis to that of the sister species X. laevis. In this system, interphase nuclei were reconstructed in vitro from sperm chromatin and increased in size throughout the incubation period. Using extracts from X. tropicalis, growth rate of the reconstructed nuclei was decreased by obstructing the effective cytoplasmic space, decreasing DNA quantity, or inhibiting molecules involved in various cytoplasmic mechanisms. Although these features are qualitatively identical to that shown by the extract of X. laevis, the sensitivities of experimental manipulation for each cellular parameter were different between the extracts from two Xenopus species. These quantitative differences implied that the contribution of each mode to expansion of the nuclear envelope is coordinated in a species‐specific manner, which sets the species‐specific nuclear size for in vivo physiological function. [ABSTRACT FROM AUTHOR]

How the embryonic genome regulates accessibility to transcription factors is one of the major questions in understanding the spatial and temporal dynamics of gene expression during embryogenesis. Epigenomic analyses of embryonic chromatin provide molecular insights into cell‐specific gene activities and genomic architectures. In recent years, significant advances have been made to elucidate the dynamic changes behind the activation of the zygotic genome in various model organisms. Here we provide an overview of the recent epigenomic studies pertaining to early Xenopus development. [ABSTRACT FROM AUTHOR]

Organogenesis is a complex process that can be disrupted by embryonic exposure to teratogens or mutation‐induced alterations in signalling pathways, both of which result in organ mispatterning. Building on prior work in Xenopus laevis that showed that increased HCN2 ion channel activity rescues nicotine‐induced brain and eye morphogenesis, we demonstrate much broader HCN2‐based rescue of organ patterning defects. Induced HCN2 expression in both local or distant tissues can rescue CNS (brain and eye) as well as non‐CNS (heart and gut) organ defects induced by three different teratogenic conditions: nicotine exposure, ethanol exposure or aberrant Notch protein. Rescue can also be induced by small‐molecule HCN2 channel activators, even with delayed treatment initiation. Our results suggest that HCN2 (likely mediated by bioelectric signals) can be an effective regulator of organogenesis from all three germ layers (ectoderm, mesoderm and endoderm) and reveal non‐cell‐autonomous influences on organ formation that work at a considerable distance during embryonic development. These results suggest molecular bioelectric strategies for repair that could be explored in the future for regenerative medicine. [ABSTRACT FROM AUTHOR]

The management of bio‐invasions relies upon the development of methods to trace their origin and expansion. Cointroduced parasites, especially monogenean flatworms, are ideal tags for the movement of their hosts due to their short generations, direct life cycles and host specificity. However, they are yet to be applied to trace the intraspecific movement of host lineages in their native ranges.As proof of this concept, we conducted a comparative phylogeographic analysis based upon two mitochondrial markers of a globally invasive frog Xenopus laevis and its monogenean parasite Protopolystoma xenopodis in its native range in southern Africa and invasive range in Europe.Translocation of lineages was largely masked in the frog's phylogeography. However, incongruent links between host and parasite phylogeography indicated host switches from one host lineage to another after these were brought into contact in the native range. Thus, past translocation of host lineages is revealed by the invasion success of its cointroduced parasite lineage.This study demonstrates that parasite data can serve as an independent line of evidence in invasion biology, also on the intraspecific level, shedding light on previously undetected invasion dynamics. Based upon the distribution of these invasive parasite lineages, we infer that there is widespread anthropogenic translocation of this frog, not only via official export routes, but also facilitated by the frog's use as live bait by angling communities.Synthesis and applications. Data from cointroduced, host‐specific parasites, as tags for translocation, can add value to investigations in invasion biology and conservation. A better understanding of the translocation history and resulting genetic mixing of host and parasite lineages in the native range can shed light on the genetic make‐up of parasite assemblages cointroduced to the invasive range. Knowledge of the intraspecific movement of different lineages of animals in their native ranges also has conservation implications, since contact between divergent lineages of hosts and parasites can facilitate host switches and altered parasite dynamics in both native and invasive populations. Therefore, we recommend the inclusion of parasite data as a more holistic approach to the invasion ecology of animals on the intraspecific level. [ABSTRACT FROM AUTHOR]

Background: Limb buds develop as bilateral outgrowths of the lateral plate mesoderm and are patterned along three axes. Current models of proximal to distal patterning of early amniote limb buds suggest that two signals, a distal organizing signal from the apical epithelial ridge (AER, Fgfs) and an opposing proximal (retinoic acid [RA]) act early on pattern this axis. Results: Transcriptional analysis of stage 51 Xenopus laevis hindlimb buds sectioned along the proximal‐distal axis showed that the distal region is distinct from the rest of the limb. Expression of capn8.3, a novel calpain, was located in cells immediately flanking the AER. The Wnt antagonist Dkk1 was AER‐specific in Xenopus limbs. Two transcription factors, sall1 and zic5, were expressed in distal mesenchyme. Zic5 has no described association with limb development. We also describe expression of two proximal genes, gata5 and tnn, not previously associated with limb development. Differentially expressed genes were associated with Fgf, Wnt, and RA signaling as well as differential cell adhesion and proliferation. Conclusions: We identify new candidate genes for early proximodistal limb patterning. Our analysis of RA‐regulated genes supports a role for transient RA gradients in early limb bud in proximal‐to‐distal patterning in this anamniote model organism. Key Findings: Transcriptome analysis revealed seven patterns of differential expression across stage 51 Xenopus hindlimb buds, with the distal region most transcriptionally distinct.Two distal (capn8.3, zic5) and two proximal (gata5 and tnn) genes with no previously described role in limb development were identified.Genes linked to Wnt, Fgf and retinoic acid (RA) signaling, cell adhesion and proliferation are differentially expressed across the proximodistal axis.All genes associated with RA signaling are proximal, and genes previously shown to be down‐regulated by RA are distal.Our findings support the presence of patterning gradients of RA across the early limb proximodistal axis in Xenopus. [ABSTRACT FROM AUTHOR]

Ventx2 is an Antennapedia superfamily/NK‐like subclass homeodomain transcription factor best known for its roles in the regulation of early dorsoventral patterning during Xenopus gastrulation and in the maintenance of neural crest multipotency. In this work we characterize the spatiotemporal expression pattern of ventx2 in progenitor cells of the Xenopus respiratory system epithelium. We find that ventx2 is directly induced by BMP signaling in the ventral foregut prior to nkx2‐1, the earliest epithelial marker of the respiratory lineage. Functional studies demonstrate that Ventx2 regulates the number of Nkx2‐1/Sox9+ respiratory progenitor cells induced during foregut development, the timing and level of surfactant protein gene expression, and proper tracheal–esophageal separation. Our data suggest that Ventx2 regulates the balance of respiratory progenitor cell expansion and differentiation. While the ventx gene family has been lost from the mouse genome during evolution, humans have retained a ventx2‐like gene (VENTX). Finally, we discuss how our findings might suggest a possible function of VENTX in human respiratory progenitor cells. [ABSTRACT FROM AUTHOR]

Observing mineralization is essential for studying skeletal development, maintenance, and regeneration. Calcein and alizarin red have long been used to visualize mineralization in fixed specimens, but this requires the target animals to be sacrificed. However, several intravital bone‐staining methods have been developed to visualize mineralized tissues in living animals. These methods have been applied to study fin rays and transparent fishes. Xenopus tropicalis is an excellent experimental animal model for studying bone formation and regeneration because skeletal mineralization begins during the free‐living tadpole period, and its regenerative ability changes during metamorphosis. However, intravital bone staining of X. tropicalis has only been reported for tadpoles, and no details on its specificity or appropriate experimental conditions are available. Here, we compared the calcein‐ and alizarin red S (ARS)‐staining methods and optimized these methods for tadpoles and juvenile frogs during and after metamorphosis. Staining with 0.01% ARS yielded acceptable signaling for young tadpoles, whereas calcein either at 0.1 or 0.01% occasionally showed artifactual staining of unmineralized tissues. In addition, 0.1% calcein or 0.1% ARS staining showed a higher signal‐to‐noise ratio with juvenile frogs compared to staining at 0.01%. We propose the use of 0.01% ARS for tadpoles before stage 61 and 0.1% ARS thereafter for staining mineralized tissues. Using this method, we found that ossification of the neural arches occurred at stage 51 in X. tropicalis. This method enables precise staging and manipulation based on the visualized bone structure. [ABSTRACT FROM AUTHOR]

We have identified a myeloid cell‐specific enhancer in the 5′ flanking region of the Xenopus tropicalis myeloperoxidase gene. Transgenic reporter analysis using Xenopus laevis revealed that the expression of GFP was detected in the tail fin macrophages of a swimming tadpole, and the distributions of the GFP‐positive and XL‐2 (a pan‐marker for leukocytes)‐positive cells were mostly overlapping. The GFP‐positive cells in the liver of the transgenic tadpole were localized in the same areas where the myeloid cells were present. Isolation of leukocytes from the peripheral blood cells followed by flow cytometric analysis revealed that the GFP‐positive fraction was specifically enriched in neutrophils with lobulated nuclei. Furthermore, the macrophages purified from the peritoneal cavity were also GFP‐positive. In summary, a transgenic frog line in which the myeloid cells are labeled with GFP provides a useful tool to elucidate the physiological role of myeloid cells of multiple origins in the embryo. [ABSTRACT FROM AUTHOR]

Early embryonic cell fates are specified through coordinated integration of transcription factor activities and epigenetic states of the genome. Foxh1 is a key maternal transcription factor controlling the mesendodermal gene regulatory program. Proteomic interactome analyses using FOXH1 as a bait in mouse embryonic stem cells revealed that FOXH1 interacts with PRC2 subunits and HDAC1. Foxh1 physically interacts with Hdac1, and confers transcriptional repression of mesendodermal genes in Xenopus ectoderm. Our findings reveal a central role of Foxh1 in coordinating the chromatin states of the Xenopus embryonic genome. [ABSTRACT FROM AUTHOR]

Background: Xenopus laevis is a widely used model organism in the fields of genetics and development, and more recently evolution. At present, the most widely used staging table for X. laevis is based primarily on external features and does not describe the corresponding skull development in detail. Here, we describe skull development in X. laevis, complete with labeled figures, for each relevant stage in the most widely used staging table. Results: We find skull development in X. laevis is, for the most part, distinct at each of the previously established stages based on external anatomy. However, variation does exist in the timing of onset of ossification of certain bones in the skull, which results in a range of stages where a skull element first ossifies. The overall sequence of ossification is less variable than the timing of ossification onset. Conclusions: While events in skull development vary somewhat between specimens, and in comparison, to external events, this staging table is useful in showing both when bones first appear and for documenting the range of temporal variance in X. laevis skull development more accurately than previously done. Furthermore, when only skull data are available, the approximate stage of a specimen can now be determined. Key Findings: We find skull development in X. laevis is, for the most part, distinct at each of the previously established stages based on external anatomy. However, variation does exist in the timing of onset of ossification of certain bones in the skull, which results in a range of stages where a skull element first ossifies. The overall sequence of ossification is less variable than the timing of ossification onset. [ABSTRACT FROM AUTHOR]