RESUMO
Covalent DNA-protein cross-links (DPCs) impede replication fork progression and threaten genome integrity. Using Xenopus egg extracts, we previously showed that replication fork collision with DPCs causes their proteolysis, followed by translesion DNA synthesis. We show here that when DPC proteolysis is blocked, the replicative DNA helicase CMG (CDC45, MCM2-7, GINS), which travels on the leading strand template, bypasses an intact leading strand DPC. Single-molecule imaging reveals that GINS does not dissociate from CMG during bypass and that CMG slows dramatically after bypass, likely due to uncoupling from the stalled leading strand. The DNA helicase RTEL1 facilitates bypass, apparently by generating single-stranded DNA beyond the DPC. The absence of RTEL1 impairs DPC proteolysis, suggesting that CMG must bypass the DPC to enable proteolysis. Our results suggest a mechanism that prevents inadvertent CMG destruction by DPC proteases, and they reveal CMG's remarkable capacity to overcome obstacles on its translocation strand.
Assuntos
DNA Helicases/metabolismo , DNA Helicases/fisiologia , Reparo do DNA/fisiologia , Animais , Proteínas de Ciclo Celular/metabolismo , DNA/metabolismo , Replicação do DNA , DNA de Cadeia Simples , Proteínas de Ligação a DNA/fisiologia , Feminino , Masculino , Proteólise , Imagem Individual de Molécula/métodos , Xenopus laevis/metabolismoRESUMO
POLθ promotes repair of DNA double-strand breaks (DSBs) resulting from collapsed forks in homologous recombination (HR) defective tumors. Inactivation of POLθ results in synthetic lethality with the loss of HR genes BRCA1/2, which induces under-replicated DNA accumulation. However, it is unclear whether POLθ-dependent DNA replication prevents HR-deficiency-associated lethality. Here, we isolated Xenopus laevis POLθ and showed that it processes stalled Okazaki fragments, directly visualized by electron microscopy, thereby suppressing ssDNA gaps accumulating on lagging strands in the absence of RAD51 and preventing fork reversal. Inhibition of POLθ DNA polymerase activity leaves fork gaps unprotected, enabling their cleavage by the MRE11-NBS1-CtIP endonuclease, which produces broken forks with asymmetric single-ended DSBs, hampering BRCA2-defective cell survival. These results reveal a POLθ-dependent genome protection function preventing stalled forks rupture and highlight possible resistance mechanisms to POLθ inhibitors.
Assuntos
Replicação do DNA , Proteínas de Ligação a DNA , Proteína Homóloga a MRE11/genética , Proteína Homóloga a MRE11/metabolismo , Proteínas de Ligação a DNA/genética , Recombinação Homóloga/genética , DNARESUMO
Cleft lip with or without cleft palate (CL/P) is a common birth defect with a complex, heterogeneous etiology. It is well established that common and rare sequence variants contribute to the formation of CL/P, but the contribution of copy-number variants (CNVs) to cleft formation remains relatively understudied. To fill this knowledge gap, we conducted a large-scale comparative analysis of genome-wide CNV profiles of 869 individuals from the Philippines and 233 individuals of European ancestry with CL/P with three primary goals: first, to evaluate whether differences in CNV number, amount of genomic content, or amount of coding genomic content existed within clefting subtypes; second, to assess whether CNVs in our cohort overlapped with known Mendelian clefting loci; and third, to identify unestablished Mendelian clefting genes. Significant differences in CNVs across cleft types or in individuals with non-syndromic versus syndromic clefts were not observed; however, several CNVs in our cohort overlapped with known syndromic and non-syndromic Mendelian clefting loci. Moreover, employing a filtering strategy relying on population genetics data that rare variants are on the whole more deleterious than common variants, we identify several CNV-associated gene losses likely driving non-syndromic clefting phenotypes. By prioritizing genes deleted at a rare frequency across multiple individuals with clefts yet enriched in our cohort of individuals with clefts compared to control subjects, we identify COBLL1, RIC1, and ARHGEF38 as clefting genes. CRISPR-Cas9 mutagenesis of these genes in Xenopus laevis and Danio rerio yielded craniofacial dysmorphologies, including clefts analogous to those seen in human clefting disorders.
Assuntos
Fenda Labial , Fissura Palatina , Variações do Número de Cópias de DNA , Humanos , Fenda Labial/genética , Fissura Palatina/genética , Estudo de Associação Genômica Ampla , Fatores de Troca do Nucleotídeo Guanina/genética , Fenótipo , Fatores de Transcrição/genéticaRESUMO
Xenopus laevis tadpoles can regenerate whole tails after amputation. We have previously reported that interleukin 11 (il11) is required for tail regeneration. In this study, we have screened for genes that support tail regeneration under Il11 signaling in a certain cell type and have identified the previously uncharacterized genes Xetrov90002578m.L and Xetrov90002579m.S [referred to hereafter as regeneration factors expressed on myeloid.L (rfem.L) and rfem.S]. Knockdown (KD) of rfem.L and rfem.S causes defects of tail regeneration, indicating that rfem.L and/or rfem.S are required for tail regeneration. Single-cell RNA sequencing (scRNA-seq) revealed that rfem.L and rfem.S are expressed in a subset of leukocytes with a macrophage-like gene expression profile. KD of colony-stimulating factor 1 (csf1), which is essential for macrophage differentiation and survival, reduced rfem.L and rfem.S expression levels and the number of rfem.L- and rfem.S-expressing cells in the regeneration bud. Furthermore, forced expression of rfem.L under control of the mpeg1 promoter, which drives rfem.L in macrophage-like cells, rescues rfem.L and rfem.S KD-induced tail regeneration defects. Our findings suggest that rfem.L or rfem.S expression in macrophage-like cells is required for tail regeneration.
Assuntos
Interleucina-11 , Transdução de Sinais , Animais , Xenopus laevis/genética , Xenopus laevis/metabolismo , Interleucina-11/metabolismo , Larva/genética , Transdução de Sinais/genética , Macrófagos , CaudaRESUMO
Loss of function variations in the dual specificity tyrosine-phosphorylation-regulated kinase 1 A (DYRK1A) gene are associated with craniofacial malformations in humans. Here we characterized the effects of deficient DYRK1A in craniofacial development using a developmental model, Xenopus laevis. Dyrk1a mRNA and protein were expressed throughout the developing head and both were enriched in the branchial arches which contribute to the face and jaw. Consistently, reduced Dyrk1a function, using dyrk1a morpholinos and pharmacological inhibitors, resulted in orofacial malformations including hypotelorism, altered mouth shape, slanted eyes, and narrower face accompanied by smaller jaw cartilage and muscle. Inhibition of Dyrk1a function resulted in misexpression of key craniofacial regulators including transcription factors and members of the retinoic acid signaling pathway. Two such regulators, sox9 and pax3 are required for neural crest development and their decreased expression corresponds with smaller neural crest domains within the branchial arches. Finally, we determined that the smaller size of the faces, jaw elements and neural crest domains in embryos deficient in Dyrk1a could be explained by increased cell death and decreased proliferation. This study is the first to provide insight into why craniofacial birth defects might arise in humans with variants of DYRK1A.
Assuntos
Quinases Dyrk , Proteínas de Xenopus , Xenopus laevis , Animais , Região Branquial/embriologia , Região Branquial/metabolismo , Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/embriologia , Anormalidades Craniofaciais/metabolismo , Embrião não Mamífero/metabolismo , Embrião não Mamífero/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Crista Neural/embriologia , Crista Neural/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Transdução de Sinais , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genéticaRESUMO
Xenopus laevis is a widely used model organism in developmental and regeneration studies. Despite several reports regarding targeted integration techniques in Xenopus, there is still room for improvement of them, especially in creating reporter lines that rely on endogenous regulatory enhancers/promoters. We developed a CRISPR-Cas9-based simple method to efficiently introduce a fluorescent protein gene into 5' untranslated regions (5'UTRs) of target genes in Xenopus laevis. A donor plasmid DNA encoding an enhanced green fluorescent protein (eGFP) flanked by a genomic fragment ranging from 66 bp to 878 bp including target 5'UTR was co-injected into fertilized eggs with a single guide RNA and Cas9 protein. Injections for krt12.2.L, myod1.S, sox2.L or brevican.S resulted in embryos expressing eGFP fluorescence in a tissue-specific manner, recapitulating endogenous expression of target genes. Integrations of the donor DNA into the target regions were examined by genotyping PCR for the eGFP-expressing embryos. The rate of embryos expressing the specific eGFP varied from 2.1% to 13.2% depending on the target locus and length of the genomic fragment in the donor plasmids. Germline transmission of an integrated DNA was observed. This simple method provides a powerful tool for exploring gene expression and function in developmental and regeneration research in X. laevis.
Assuntos
Sistemas CRISPR-Cas , RNA Guia de Sistemas CRISPR-Cas , Animais , Xenopus laevis/genética , Sistemas CRISPR-Cas/genética , Expressão Gênica , DNARESUMO
Normal tables of development are essential for studies of embryogenesis, serving as an important resource for model organisms, including the frog Xenopus laevis. Xenopus has long been used to study developmental and cell biology, and is an increasingly important model for human birth defects and disease, genomics, proteomics and toxicology. Scientists utilize Nieuwkoop and Faber's classic 'Normal Table of Xenopus laevis (Daudin)' and accompanying illustrations to enable experimental reproducibility and reuse the illustrations in new publications and teaching. However, it is no longer possible to obtain permission for these copyrighted illustrations. We present 133 new, high-quality illustrations of X. laevis development from fertilization to metamorphosis, with additional views that were not available in the original collection. All the images are available on Xenbase, the Xenopus knowledgebase (http://www.xenbase.org/entry/zahn.do), for download and reuse under an attributable, non-commercial creative commons license. Additionally, we have compiled a 'Landmarks Table' of key morphological features and marker gene expression that can be used to distinguish stages quickly and reliably (https://www.xenbase.org/entry/landmarks-table.do). This new open-access resource will facilitate Xenopus research and teaching in the decades to come.
Assuntos
Bases de Dados Genéticas , Genômica , Animais , Humanos , Metamorfose Biológica , Reprodutibilidade dos Testes , Xenopus laevis/genéticaRESUMO
Oocyte maturation and early embryo development occur in vertebrates in the near absence of transcription. Thus, sexual reproduction of vertebrates critically depends on the timely translation of mRNAs already stockpiled in the oocyte. Yet how translational activation of specific mRNAs is temporally coordinated is still incompletely understood. Here, we elucidate the function of Zar1l, a yet uncharacterized member of the Zar RNA-binding protein family, in Xenopus oocytes. Employing TRIM-Away, we demonstrate that loss of Zar1l accelerates hormone-induced meiotic resumption of Xenopus oocytes due to premature accumulation of the M-phase-promoting kinase cMos. We show that Zar1l is a constituent of a large ribonucleoparticle containing the translation repressor 4E-T and the central polyadenylation regulator CPEB1, and that it binds directly to the cMos mRNA. Partial, hormone-induced degradation of Zar1l liberates 4E-T from CPEB1, which weakens translational repression of mRNAs encoding cMos and likely additional M-phase-promoting factors. Thus, our study provides fundamental insights into the mechanisms that ensure temporally regulated translation of key cell cycle regulators during oocyte maturation, which is essential for sexual reproductivity.
Assuntos
Meiose , Oócitos , Animais , Xenopus laevis/genética , Xenopus laevis/metabolismo , Oócitos/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Hormônios/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Biossíntese de ProteínasRESUMO
Brca2 deficiency causes Mre11-dependent degradation of nascent DNA at stalled forks, leading to cell lethality. To understand the molecular mechanisms underlying this process, we isolated Xenopus laevis Brca2. We demonstrated that Brca2 protein prevents single-stranded DNA gap accumulation at replication fork junctions and behind them by promoting Rad51 binding to replicating DNA. Without Brca2, forks with persistent gaps are converted by Smarcal1 into reversed forks, triggering extensive Mre11-dependent nascent DNA degradation. Stable Rad51 nucleofilaments, but not RPA or Rad51T131P mutant proteins, directly prevent Mre11-dependent DNA degradation. Mre11 inhibition instead promotes reversed fork accumulation in the absence of Brca2. Rad51 directly interacts with the Pol α N-terminal domain, promoting Pol α and δ binding to stalled replication forks. This interaction likely promotes replication fork restart and gap avoidance. These results indicate that Brca2 and Rad51 prevent formation of abnormal DNA replication intermediates, whose processing by Smarcal1 and Mre11 predisposes to genome instability.
Assuntos
Proteína BRCA2/metabolismo , Replicação do DNA , DNA/biossíntese , Rad51 Recombinase/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , Proteína BRCA2/genética , Sítios de Ligação , DNA/genética , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Polimerase I/metabolismo , DNA Polimerase III/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Feminino , Instabilidade Genômica , Humanos , Proteína Homóloga a MRE11 , Masculino , Mutação , Ligação Proteica , Rad51 Recombinase/genética , Origem de Replicação , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Tempo , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMO
Lysosomes are the digestive center of the cell and play important roles in human diseases, including cancer. Previous work has suggested that late endosomes, also known as multivesicular bodies (MVBs), and lysosomes are essential for canonical Wnt pathway signaling. Sequestration of Glycogen Synthase 3 (GSK3) and of ßcatenin destruction complex components in MVBs is required for sustained canonical Wnt signaling. Little is known about the role of lysosomes during early development. In the Xenopus egg, a Wnt-like cytoplasmic determinant signal initiates formation of the body axis following a cortical rotation triggered by sperm entry. Here we report that cathepsin D was activated in lysosomes specifically on the dorsal marginal zone of the embryo at the 64-cell stage, long before zygotic transcription starts. Expansion of the MVB compartment with low-dose hydroxychloroquine (HCQ) greatly potentiated the dorsalizing effects of the Wnt agonist lithium chloride (LiCl) in embryos, and this effect required macropinocytosis. Formation of the dorsal axis required lysosomes, as indicated by brief treatments with the vacuolar ATPase (V-ATPase) inhibitors Bafilomycin A1 or Concanamycin A at the 32-cell stage. Inhibiting the MVB-forming machinery with a dominant-negative point mutation in Vacuolar Protein Sorting 4 (Vps4-EQ) interfered with the endogenous dorsal axis. The Wnt-like activity of the dorsal cytoplasmic determinant Huluwa (Hwa), and that of microinjected xWnt8 messenger RNA, also required lysosome acidification and the MVB-forming machinery. We conclude that lysosome function is required for early dorsal axis development in Xenopus. The results highlight the intertwining between membrane trafficking, lysosomes, and vertebrate axis formation.
Assuntos
Lisossomos , Transdução de Sinais , Animais , Padronização Corporal , Embrião de Mamíferos , Embrião não Mamífero , Proteínas de Xenopus , Xenopus laevisRESUMO
The development of functional topography in the developing brain follows a progression from initially coarse to more precisely organized maps. To examine the emergence of topographically organized maps in the retinotectal system, we performed longitudinal visual receptive field mapping by calcium imaging in the optic tectum of GCaMP6-expressing transgenic Xenopus laevis tadpoles. At stage 42, just 1 d after retinal axons arrived in the optic tectum, a clear retinotopic azimuth map was evident. Animals were imaged over the following week at stages 45 and 48, over which time the tectal neuropil nearly doubled in length and exhibited more precise retinotopic organization. By microinjecting GCaMP6s messenger ribonucleic acid (mRNA) into one blastomere of two-cell stage embryos, we acquired bilateral mosaic tadpoles with GCaMP6s expression in postsynaptic tectal neurons on one side of the animal and in retinal ganglion cell axons crossing to the tectum on the opposite side. Longitudinal observation of retinotopic map emergence revealed the presence of orderly representations of azimuth and elevation as early as stage 42, although presynaptic inputs exhibited relatively less topographic organization than the postsynaptic component for the azimuth axis. Retinotopic gradients in the tectum became smoother between stages 42 and 45. Blocking N-methyl-D-aspartate (NMDA) receptor conductance by rearing tadpoles in MK-801 did not prevent the emergence of retinotopic maps, but it produced more discontinuous topographic gradients and altered receptive field characteristics. These results provide evidence that current through NMDA receptors is dispensable for coarse topographic ordering of retinotectal inputs but does contribute to the fine-scale organization of the retinotectal projection.
Assuntos
Receptores de N-Metil-D-Aspartato/metabolismo , Retina/diagnóstico por imagem , Retina/embriologia , Animais , Axônios/metabolismo , Mapeamento Encefálico/métodos , Cálcio/metabolismo , Larva/metabolismo , Células Ganglionares da Retina/fisiologia , Colículos Superiores/diagnóstico por imagem , Colículos Superiores/metabolismo , Vias Visuais/crescimento & desenvolvimento , Xenopus laevis/embriologiaRESUMO
Despite the recent discovery of tissue regeneration enhancers in highly regenerative animals, upstream and downstream genetic programs connected by these enhancers still remain unclear. Here, we performed a genome-wide analysis of enhancers and associated genes in regenerating nephric tubules of Xenopus laevis. Putative enhancers were identified using assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) analyses. Their target genes were predicted based on their proximity to enhancers on genomic DNA and consistency of their transcriptome profiles to ATAC-seq/ChIP-seq profiles of the enhancers. Motif enrichment analysis identified the central role of Krüppel-like factors (Klf) in the enhancer. Klf15, a member of the Klf family, directly binds enhancers and stimulates expression of regenerative genes, including adrenoreceptor alpha 1A (adra1a), whereas inhibition of Klf15 activity results in failure of nephric tubule regeneration. Moreover, pharmacological inhibition of Adra1a-signaling suppresses nephric tubule regeneration, while its activation promotes nephric tubule regeneration and restores organ size. These results indicate that Klf15-dependent adrenergic receptor signaling through regeneration enhancers plays a central role in the genetic network for kidney regeneration.
Assuntos
Elementos Facilitadores Genéticos , Túbulos Renais , Fatores de Transcrição Kruppel-Like , Receptores Adrenérgicos , Regeneração , Animais , Cromatina/metabolismo , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Túbulos Renais/fisiologia , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Receptores Adrenérgicos/metabolismo , Regeneração/genética , Xenopus laevisRESUMO
Xenopus oocytes are encompassed by a layer of follicular cells that contribute to oocyte growth and meiosis in relation to oocyte maturation. However, the effects of the interaction between follicular cells and the oocyte surface on meiotic processes are unclear. Here, we investigated Xenopus follicular cell function using oocyte signaling and heterologous-expressing capabilities. We found that oocytes deprotected from their surrounding layer of follicular cells and expressing the epidermal growth factor (EGF) receptor (EGFR) and the Grb7 adaptor undergo accelerated prophase I to metaphase II meiosis progression upon stimulation by EGF. This unusual maturation unravels atypical spindle formation but is rescued by inhibiting integrin ß1 or Grb7 binding to the EGFR. In addition, we determined that oocytes surrounded by their follicular cells expressing EGFR-Grb7 exhibit normal meiotic resumption. These oocytes are protected from abnormal meiotic spindle formation through the recruitment of O-GlcNAcylated Grb7, and OGT (O-GlcNAc transferase), the enzyme responsible for O-GlcNAcylation processes, in the integrin ß1-EGFR complex. Folliculated oocytes can be forced to adopt an abnormal phenotype and exclusive Grb7 Y338 and Y188 phosphorylation instead of O-GlcNAcylation under integrin activation. Furthermore, an O-GlcNAcylation increase (by inhibition of O-GlcNAcase), the glycosidase that removes O-GlcNAc moieties, or decrease (by inhibition of OGT) amplifies oocyte spindle defects when follicular cells are absent highlighting a control of the meiotic spindle by the OGT-O-GlcNAcase duo. In summary, our study provides further insight into the role of the follicular cell layer in oocyte meiosis progression.
Assuntos
Fator de Crescimento Epidérmico , Integrina beta1 , Oócitos , Xenopus laevis , Animais , Acilação , Regulação para Baixo , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Proteína Adaptadora GRB7/metabolismo , Integrina beta1/genética , Integrina beta1/metabolismo , Meiose , Oócitos/citologia , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Fuso Acromático/metabolismo , Xenopus laevis/metabolismoRESUMO
Regenerative abilities are not evenly distributed across the animal kingdom. The underlying modalities are also highly variable. Retinal repair can involve the mobilization of different cellular sources, including ciliary marginal zone (CMZ) stem cells, the retinal pigmented epithelium (RPE), or Müller glia. To investigate whether the magnitude of retinal damage influences the regeneration modality of the Xenopus retina, we developed a model based on cobalt chloride (CoCl2 ) intraocular injection, allowing for a dose-dependent control of cell death extent. Analyses in Xenopus laevis revealed that limited CoCl2 -mediated neurotoxicity only triggers cone loss and results in a few Müller cells reentering the cell cycle. Severe CoCl2 -induced retinal degeneration not only potentializes Müller cell proliferation but also enhances CMZ activity and unexpectedly triggers RPE reprogramming. Surprisingly, reprogrammed RPE self-organizes into an ectopic mini-retina-like structure laid on top of the original retina. It is thus likely that the injury paradigm determines the awakening of different stem-like cell populations. We further show that these cellular sources exhibit distinct neurogenic capacities without any bias towards lost cells. This is particularly striking for Müller glia, which regenerates several types of neurons, but not cones, the most affected cell type. Finally, we found that X. tropicalis also has the ability to recruit Müller cells and reprogram its RPE following CoCl2 -induced damage, whereas only CMZ involvement was reported in previously examined degenerative models. Altogether, these findings highlight the critical role of the injury paradigm and reveal that three cellular sources can be reactivated in the very same degenerative model.
Assuntos
Cobalto , Degeneração Retiniana , Animais , Xenopus laevis/fisiologia , Degeneração Retiniana/induzido quimicamente , Degeneração Retiniana/metabolismo , Retina , Regeneração/fisiologia , Proliferação de Células , Neuroglia/metabolismoRESUMO
Ibuprofen, one of the most commonly prescribed nonsteroidal anti-inflammatory drugs, has not been fully assessed for embryonic toxicity in vertebrates. Here, we systematically assessed the embryotoxicity of ibuprofen in Xenopus laevis at various concentrations during embryogenesis. Embryos were treated with different concentrations of ibuprofen, ranging from 8 to 64 mg/L, at 23 °C for 96 h, and examined daily and evaluated at 72 hpf. Lethal or teratogenic effects were documented. For histological analysis, paraffin embedded embryos were transversely sectioned at a thickness of 10-µm and stained with hematoxylin and eosin. Total RNA was isolated from embryos at stages 6, 12, 22 and 36, and real-time quantitative PCR was performed. Ibuprofen-treated embryos showed delayed or failed dorsal lip formation and its closure at the beginning of gastrulation. This resulted in herniation of the endodermal mass after gastrulation under high concentrations of ibuprofen-treated embryos. Underdeveloped intestines with stage and/or intestinal malrotation, distorted microcephaly, and hypoplastic heart, lungs, and pronephric tubules were observed in ibuprofen-treated embryos. Cephalic, cardiac, and truncal edema were also observed in them. The severity of the deformities was observed in a concentration-dependent manner. The teratogenic index was 2.28. These gross and histological disruptions correlated well with the altered expression of each organ marker gene. In conclusion, ibuprofen induced delayed and disrupted gastrulation in the early developmental stage and multiorgan malformation later in the organogenesis stage of Xenopus laevis embryos.
Assuntos
Ibuprofeno , Teratogênicos , Animais , Xenopus laevis , Ibuprofeno/toxicidade , Desenvolvimento Embrionário , Anti-Inflamatórios não Esteroides/farmacologia , Embrião não MamíferoRESUMO
Rab11 family-interacting protein 5 (Rab11fip5) is an adaptor protein that binds to the small GTPase Rab11, which has an important function in endosome recycling and trafficking of cellular proteins to the plasma membrane. Rab11fip5 is involved in many cellular processes, such as cytoskeleton rearrangement, iron uptake and exocytosis in neuroendocrine cells, and is also known as a candidate gene for autism-spectrum disorder. However, the role of Rab11fip5 during early embryonic development is not clearly understood. In this study, we identified Rab11fip5 as a protein that interacts with ephrinB1, a transmembrane ligand for Eph receptors. The PDZ binding motif in ephrinB1 and the Rab-binding domain in Rab11fip5 are necessary for their interaction in a complex. EphrinB1 and Rab11fip5 display overlapping expression in the telencephalon of developing amphibian embryos. The loss of Rab11fip5 function causes a reduction in telencephalon size and a decrease in the expression level of ephrinB1. Moreover, morpholino oligonucleotide-mediated knockdown of Rab11fip5 decreases cell proliferation in the telencephalon. The overexpression of ephrinB1 rescues these defects, suggesting that ephrinB1 recycling by the Rab11/Rab11fip5 complex is crucial for proper telencephalon development.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Efrina-B1/metabolismo , Telencéfalo/crescimento & desenvolvimento , Telencéfalo/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proliferação de Células , Citoesqueleto , Endossomos/metabolismo , Efrina-B1/genética , Exocitose , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Neurogênese , Telencéfalo/citologia , Xenopus laevisRESUMO
We previously identified Xenopus tudor domain containing 6/Xenopus tudor repeat (Xtdrd6/Xtr), which was exclusively expressed in the germ cells of adult Xenopus laevis. Western blot analysis showed that the XTdrd6/Xtr protein was translated in St. I/II oocytes and persisted as a maternal factor until the tailbud stage. XTdrd6/Xtr has been reported to be essential for the translation of maternal mRNA involved in oocyte meiosis. In the present study, we examined the distribution of the XTdrd6/Xtr protein during oogenesis and early development, to predict the time point of its action during development. First, we showed that XTdrd6/Xtr is localized to germinal granules in the germplasm by electron microscopy. XTdrd6/Xtr was found to be localized to the origin of the germplasm, the mitochondrial cloud of St. I oocytes, during oogenesis. Notably, XTdrd6/Xtr was also found to be localized around the nuclear membrane of St. I oocytes. This suggests that XTdrd6/Xtr may immediately interact with some mRNAs that emerge from the nucleus and translocate to the mitochondrial cloud. XTdrd6/Xtr was also detected in primordial germ cells and germ cells throughout development. Using transgenic Xenopus expressing XTdrd6/Xtr with a C-terminal FLAG tag produced by homology-directed repair, we found that the zygotic translation of the XTdrd6/Xtr protein began at St. 47/48. As germ cells are surrounded by gonadal somatic cells and are considered to enter a new differentiation stage at this phase, the newly synthesized XTdrd6/Xtr protein may regulate the translation of mRNAs involved in the new steps of germ cell differentiation.
Assuntos
Células Germinativas , Gônadas , Mesoderma , Proteínas de Xenopus , Animais , Células Germinativas/metabolismo , Gônadas/embriologia , Oócitos , Oogênese/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Xenopus laevis/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMO
Understanding how living tissues respond to changes in their mechanical environment is a key question in evolutionary biology. Invasive species provide an ideal model for this as they are often transplanted between environments that differ drastically in their ecological and environmental context. Spatial sorting, the name given to the phenomenon driving differences between individuals at the core and edge of an expanding range, has been demonstrated to impact the morphology and physiology of Xenopus laevis from the invasive French population. Here, we combined a structural analysis using micro-CT scanning and a functional analysis by testing the mechanical properties of the femur to test whether the increased dispersal at the range edge drives differences in bone morphology and function. Our results show significant differences in the inner structure of the femur as well as bone material properties, with frogs from the centre of the range having more robust and resistant bones. This is suggestive of an energy allocation trade-off between locomotion and investment in bone formation, or alternatively, may point to selection for fast locomotion at the range edge. Overall, our results provide insights on the growth of the long bones and the formation of trabecular bone in frogs.
Assuntos
Fêmur , Espécies Introduzidas , Microtomografia por Raio-X , Xenopus laevis , Animais , Xenopus laevis/fisiologia , Xenopus laevis/anatomia & histologia , Xenopus laevis/crescimento & desenvolvimento , Fêmur/fisiologia , Fêmur/anatomia & histologia , Fenômenos Biomecânicos , Locomoção/fisiologia , França , FemininoRESUMO
Cohesion between sister chromatids by the cohesin protein complex ensures accurate chromosome segregation and enables recombinational DNA repair. Sister chromatid cohesion is promoted by acetylation of the SMC3 subunit of cohesin by the ESCO2 acetyltransferase, inhibiting cohesin release from chromatin. The interaction of ESCO2 with the DNA replication machinery, in part through PCNA-interacting protein (PIP) motifs in ESCO2, is required for full cohesion establishment. Recent reports have suggested that Cul4-dependent degradation regulates the level of ESCO2 protein following replication. To follow up on these observations, we have characterized ESCO2 stability in Xenopus egg extracts, a cell-free system that recapitulates cohesion establishment in vitro. We found that ESCO2 was stable during DNA replication in this system. Indeed, further challenging the system by inducing DNA damage signaling or increasing the number of nuclei undergoing DNA replication had no significant impact on the stability of ESCO2. In transgenic somatic cell lines, we also did not see evidence of GFP-ESCO2 degradation during S phase of the cell cycle using both flow cytometry and live-cell imaging. We conclude that ESCO2 is stable during DNA replication in both embryonic and somatic cells.
Assuntos
Proteínas de Ciclo Celular , Replicação do DNA , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Acetiltransferases/metabolismo , CoesinasRESUMO
Ionizing radiation (IR) poses a significant threat to both the natural environment and biological health. Exposure to specific doses of ionizing radiation early in an organism's development can lead to developmental toxicity, particularly neurotoxicity. Through experimentation with Xenopus laevis (X. laevis), we examined the effects of radiation on early developmental stage. Our findings revealed that radiation led to developmental abnormalities and mortality in X. laevis embryos in a dose-dependent manner, disrupting redox homeostasis and inducing cell apoptosis. Additionally, radiation caused neurotoxic effects, resulting in abnormal behavior and neuron damage in the embryos. Further investigation into the underlying mechanisms of radiation-induced neurotoxicity indicated the potential involvement of the neuroactive ligand-receptor interaction pathway, which was supported by RNA-Seq analysis. Validation of gene expression associated with this pathway and analysis of neurotransmitter levels confirmed our hypothesis. In addition, we further validated the important role of this signaling pathway in radiation-induced neurotoxicity through edaravone rescue experiments. This research establishes a valuable model for radiation damage studying and provides some insight into radiation-induced neurotoxicity mechanisms.