RESUMO
Investigating cell lineage requires genetic tools that label cells in a temporal and tissue-specific manner. The bacteriophage-derived Cre-ERT2 /loxP system has been developed as a genetic tool for lineage tracing in many organisms. We recently reported a stable transgenic Xenopus line with a Cre-ERT2 /loxP system driven by the mouse Prrx1 (mPrrx1) enhancer to trace limb fibroblasts during the regeneration process (Prrx1:CreER line). Here we describe the detailed technological development and characterization of such line. Transgenic lines carrying a CAG promoter-driven Cre-ERT2 /loxP system showed conditional labeling of muscle, epidermal, and interstitial cells in both the tadpole tail and the froglet leg upon 4-hydroxytamoxifen (4OHT) treatment. We further improved the labeling efficiency in the Prrx1:CreER lines from 12.0% to 32.9% using the optimized 4OHT treatment regime. Careful histological examination showed that Prrx1:CreER lines also sparsely labeled cells in the brain, spinal cord, head dermis, and fibroblasts in the tail. This work provides the first demonstration of conditional, tissue-specific cell labeling with the Cre-ERT2 /loxP system in stable transgenic Xenopus lines.
Assuntos
Integrases , Animais , Animais Geneticamente Modificados , Integrases/genética , Integrases/metabolismo , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
Protein purification is the vital basis to study the function, structure and interaction of proteins. Widely used methods are affinity chromatography-based purifications, which require different chromatography columns and harsh conditions, such as acidic pH and/or adding imidazole or high salt concentration, to elute and collect the purified proteins. Here we established an easy and fast purification method for soluble proteins under mild conditions, based on the light-induced protein dimerization system improved light-induced dimer (iLID), which regulates protein binding and release with light. We utilize the biological membrane, which can be easily separated by centrifugation, as the port to anchor the target proteins. In Xenopus laevis oocyte and Escherichia coli, the blue light-sensitive part of iLID, AsLOV2-SsrA, was targeted to the plasma membrane by different membrane anchors. The other part of iLID, SspB, was fused with the protein of interest (POI) and expressed in the cytosol. The SspB-POI can be captured to the membrane fraction through light-induced binding to AsLOV2-SsrA and then released purely to fresh buffer in the dark after simple centrifugation and washing. This method, named mem-iLID, is very flexible in scale and economic. We demonstrate the quickly obtained yield of two pure and fully functional enzymes: a DNA polymerase and a light-activated adenylyl cyclase. Furthermore, we also designed a new SspB mutant for better dissociation and less interference with the POI, which could potentially facilitate other optogenetic manipulations of protein-protein interaction.
Assuntos
Adenilil Ciclases/isolamento & purificação , DNA Polimerase Dirigida por DNA/isolamento & purificação , Proteínas de Escherichia coli/isolamento & purificação , Optogenética , Engenharia de Proteínas , Proteínas de Xenopus/isolamento & purificação , Adenilil Ciclases/genética , Animais , Membrana Celular/enzimologia , Membrana Celular/genética , Análise Custo-Benefício , DNA Polimerase Dirigida por DNA/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Luz , Mutação , Optogenética/economia , Ligação Proteica , Engenharia de Proteínas/economia , Multimerização Proteica , Proteínas Recombinantes de Fusão/isolamento & purificação , Fatores de Tempo , Fluxo de Trabalho , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
We previously identified a deletion on chromosome 16p12.1 that is mostly inherited and associated with multiple neurodevelopmental outcomes, where severely affected probands carried an excess of rare pathogenic variants compared to mildly affected carrier parents. We hypothesized that the 16p12.1 deletion sensitizes the genome for disease, while "second-hits" in the genetic background modulate the phenotypic trajectory. To test this model, we examined how neurodevelopmental defects conferred by knockdown of individual 16p12.1 homologs are modulated by simultaneous knockdown of homologs of "second-hit" genes in Drosophila melanogaster and Xenopus laevis. We observed that knockdown of 16p12.1 homologs affect multiple phenotypic domains, leading to delayed developmental timing, seizure susceptibility, brain alterations, abnormal dendrite and axonal morphology, and cellular proliferation defects. Compared to genes within the 16p11.2 deletion, which has higher de novo occurrence, 16p12.1 homologs were less likely to interact with each other in Drosophila models or a human brain-specific interaction network, suggesting that interactions with "second-hit" genes may confer higher impact towards neurodevelopmental phenotypes. Assessment of 212 pairwise interactions in Drosophila between 16p12.1 homologs and 76 homologs of patient-specific "second-hit" genes (such as ARID1B and CACNA1A), genes within neurodevelopmental pathways (such as PTEN and UBE3A), and transcriptomic targets (such as DSCAM and TRRAP) identified genetic interactions in 63% of the tested pairs. In 11 out of 15 families, patient-specific "second-hits" enhanced or suppressed the phenotypic effects of one or many 16p12.1 homologs in 32/96 pairwise combinations tested. In fact, homologs of SETD5 synergistically interacted with homologs of MOSMO in both Drosophila and X. laevis, leading to modified cellular and brain phenotypes, as well as axon outgrowth defects that were not observed with knockdown of either individual homolog. Our results suggest that several 16p12.1 genes sensitize the genome towards neurodevelopmental defects, and complex interactions with "second-hit" genes determine the ultimate phenotypic manifestation.
Assuntos
Encéfalo/metabolismo , Deleção Cromossômica , Cromossomos Humanos Par 16/genética , Transtornos do Neurodesenvolvimento/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Encéfalo/patologia , Canais de Cálcio/genética , Moléculas de Adesão Celular/genética , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Epistasia Genética/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Metiltransferases/genética , Transtornos do Neurodesenvolvimento/patologia , Proteínas Nucleares/genética , PTEN Fosfo-Hidrolase/genética , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMO
Failure to predict drug-induced toxicity reactions is a major problem contributing to a high attrition rate and tremendous cost in drug development. Drug screening in X. laevis embryos is high-throughput relative to screening in rodents, potentially making them ideal for this use. Xenopus embryos have been used as a toxicity model in the frog embryo teratogenesis assay on Xenopus (FETAX) for the early stages of drug safety evaluation. We previously developed compound-screening methods using Xenopus embryos and believe they could be used for in vitro drug-induced toxicity safety assessment before expensive preclinical trials in mammals. Specifically, Xenopus embryos could help predict drug-induced hepatotoxicity and consequently aid lead candidate prioritization. Here we present methods, which we have modified for use on Xenopus embryos, to help measure the potential for a drug to induce liver toxicity. One such method examines the release of the liver-specific microRNA (miRNA) miR-122 from the liver into the vasculature as a result of hepatocellular damage, which could be due to drug-induced acute liver injury. Paracetamol, a known hepatotoxin at high doses, can be used as a positive control. We previously showed that some of the phenotypes of mammalian paracetamol overdose are reflected in Xenopus embryos. Consequently, we have also included here a method that measures the concentration of free glutathione (GSH), which is an indicator of paracetamol-induced liver injury. These methods can be used as part of a panel of protocols to help predict the hepatoxicity of a drug at an early stage in drug development.
Assuntos
Anormalidades Induzidas por Medicamentos/diagnóstico , Bioensaio/métodos , Doença Hepática Induzida por Substâncias e Drogas/diagnóstico , Embrião não Mamífero/efeitos dos fármacos , Xenopus laevis/embriologia , Anormalidades Induzidas por Medicamentos/genética , Anormalidades Induzidas por Medicamentos/metabolismo , Animais , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Embrião não Mamífero/metabolismo , Fígado/efeitos dos fármacos , Fígado/embriologia , Fígado/metabolismo , MicroRNAs/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Testes de Toxicidade/métodos , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
Genes of the major histocompatibility complex (MHC) that underlie the adaptive immune system may allow vertebrates to recognize their kin. True kin-recognition genes should produce signalling products to which organisms can respond. Allelic variation in the peptide-binding region (PBR) of MHC molecules determines the pool of peptides that can be presented to trigger an immune response. To examine whether these MHC peptides also might underlie assessments of genetic similarity, we tested whether Xenopus laevis tadpoles socially discriminate between pairs of siblings with which they differed in PBR amino acid sequences. We found that tadpoles (four sibships, n = 854) associated preferentially with siblings with which they were more similar in PBR amino acid sequence. Moreover, the strength of their preference for a conspecific was directly proportional to the sequence similarity between them. Discrimination was graded, and correlated more closely with functional sequence differences encoded by MHC class I and class II alleles than with numbers of shared haplotypes. Our results thus suggest that haplotype analyses may fail to reveal fine-scale behavioural responses to divergence in functionally expressed sequences. We conclude that MHC-PBR gene products mediate quantitative social assessment of immunogenetic similarity that may facilitate kin recognition in vertebrates.
Assuntos
Complexo Principal de Histocompatibilidade/genética , Discriminação Social , Xenopus laevis/fisiologia , Animais , Haplótipos , Larva/genética , Larva/imunologia , Larva/fisiologia , Reação em Cadeia da Polimerase , Polimorfismo Genético , Análise de Sequência de Proteína , Xenopus laevis/genética , Xenopus laevis/imunologiaRESUMO
The Medicago truncatula NIP/LATD (for Numerous Infections and Polyphenolics/Lateral root-organ Defective) gene encodes a protein found in a clade of nitrate transporters within the large NRT1(PTR) family that also encodes transporters of dipeptides and tripeptides, dicarboxylates, auxin, and abscisic acid. Of the NRT1(PTR) members known to transport nitrate, most are low-affinity transporters. Here, we show that M. truncatula nip/latd mutants are more defective in their lateral root responses to nitrate provided at low (250 µm) concentrations than at higher (5 mm) concentrations; however, nitrate uptake experiments showed no discernible differences in uptake in the mutants. Heterologous expression experiments showed that MtNIP/LATD encodes a nitrate transporter: expression in Xenopus laevis oocytes conferred upon the oocytes the ability to take up nitrate from the medium with high affinity, and expression of MtNIP/LATD in an Arabidopsis chl1(nrt1.1) mutant rescued the chlorate susceptibility phenotype. X. laevis oocytes expressing mutant Mtnip-1 and Mtlatd were unable to take up nitrate from the medium, but oocytes expressing the less severe Mtnip-3 allele were proficient in nitrate transport. M. truncatula nip/latd mutants have pleiotropic defects in nodulation and root architecture. Expression of the Arabidopsis NRT1.1 gene in mutant Mtnip-1 roots partially rescued Mtnip-1 for root architecture defects but not for nodulation defects. This suggests that the spectrum of activities inherent in AtNRT1.1 is different from that possessed by MtNIP/LATD, but it could also reflect stability differences of each protein in M. truncatula. Collectively, the data show that MtNIP/LATD is a high-affinity nitrate transporter and suggest that it could have another function.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Genes de Plantas , Medicago truncatula/metabolismo , Nitratos/metabolismo , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Alelos , Animais , Proteínas de Transporte de Ânions/genética , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Transporte Biológico , Cloratos/metabolismo , Cloratos/farmacologia , Teste de Complementação Genética , Medicago truncatula/efeitos dos fármacos , Medicago truncatula/genética , Medicago truncatula/microbiologia , Transportadores de Nitrato , Nitratos/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Fenótipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Nodulação , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Compostos de Potássio/farmacologia , Estabilidade Proteica , Sinorhizobium meliloti/crescimento & desenvolvimento , Simbiose , Espectrometria de Massas em Tandem , Transformação Genética , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
The Xenopus inner ear provides a useful model for studies of hearing and balance because it shares features with the mammalian inner ear, and because amphibians are capable of regenerating damaged mechanosensory hair cells. The structure and function of many proteins necessary for inner ear function have yet to be elucidated and require methods for analysis. To this end, we seek to characterize Xenopus inner ear genes outside of the animal model through heterologous expression in cell lines. As part of this effort, we aimed to optimize physical (electroporation), chemical (lipid-mediated; Lipofectamine™ 2000, Metafectene® Pro), and biological (viral-mediated; BacMam virus Cellular Lights™ Tubulin-RFP) gene delivery methods in amphibian (Xenopus; A6) cells and mammalian (Chinese hamster ovary (CHO)) cells. We successfully introduced the commercially available pEGFP-N3, pmCherry-N1, pEYFP-Tubulin, and Cellular Lights™ Tubulin-RFP fluorescent constructs to cells and evaluated their transfection or transduction efficiencies using the three gene delivery methods. In addition, we analyzed the transfection efficiency of a novel construct synthesized in our laboratory by cloning the Xenopus inner ear calcium-activated potassium channel ß1 subunit, then subcloning the subunit into the pmCherry-N1 vector. Every gene delivery method was significantly more effective in CHO cells. Although results for the A6 cell line were not statistically significant, both cell lines illustrate a trend towards more efficient gene delivery using viral-mediated methods; however the cost of viral transduction is also much higher. Our findings demonstrate the need to improve gene delivery methods for amphibian cells and underscore the necessity for a greater understanding of amphibian cell biology.
Assuntos
Orelha Interna/metabolismo , Expressão Gênica , Técnicas de Transferência de Genes , Rim/citologia , Animais , Células CHO , Custos e Análise de Custo , Cricetinae , Cricetulus , Eletroporação , Técnicas de Transferência de Genes/economia , Lipídeos/química , Fatores de Tempo , Transdução Genética , Transfecção , Xenopus laevis/genéticaRESUMO
The current study describes the effect of cypermethrin, fluoxetine, and thiabendazole, at environmentally relevant concentrations, on the expression of heat shock protein 70 (HSP70) and interleukin 1ß (IL-1ß), using Xenopus laevis larvae as animal model. Cytokines and interleukins are considered good predictors of the immunotoxic potential of xenobiotics. Tadpoles at stage 47 (normal tables of X. laevis) were exposed under static conditions to: 0.3 and 30 µg/L fluoxetine, 0.7 µg/L thiabendazole, and 0.24 µg/L cypermethrin. The effects were evaluated at 7, 24, and 72 h, and 6 and 9 d. Randomly chosen tadpoles were used as genetic material for detection of hsp70 and IL-1ß mRNA induction through reverse transcription PCR. Tadpoles exposed to 30 µg/L fluoxetine showed mRNA expression of both genes at all exposure times, whereas at 0.3 µg/L a peak response for hsp70 was observed after 24 h, and the increase in IL-1ß mRNA was statistically significant with respect to the control 72 h after exposure. Thiabendazole induced a high expression of mRNA for both hsp70 and IL-1ß at all exposure times. Cypermethrin increased the hsp70 mRNA levels, with a peak at 24 h, and provoked high expression of IL-1ß mRNA at all exposure times. Considering the relationship between HSP70 and IL-1ß and their involvement (mainly of IL-1ß) in immune responses, certain changes observed in their expression could be considered warning indicators of potential immunotoxic effects of these substances on Xenopus.
Assuntos
Proteínas de Choque Térmico HSP70/genética , Imunotoxinas/toxicidade , Interleucina-1beta/genética , Poluentes Químicos da Água/toxicidade , Xenopus laevis/genética , Xenopus laevis/imunologia , Animais , Calibragem , Fluoxetina/análise , Fluoxetina/toxicidade , Imunotoxinas/análise , Piretrinas/análise , Piretrinas/toxicidade , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tiabendazol/análise , Tiabendazol/toxicidade , Poluentes Químicos da Água/análiseRESUMO
The SRY-related, HMG box SoxB1 transcription factors are highly homologous, evolutionarily conserved proteins that are expressed in neuroepithelial cells throughout neural development. SoxB1 genes are down-regulated as cells exit the cell-cycle to differentiate and are considered functionally redundant in maintaining neural precursor populations. However, little is known about Sox3 function and its mode of action during primary neurogenesis. Using gain and loss-of-function studies, we analyzed Sox3 function in detail in Xenopus early neural development and compared it to that of Sox2. Through these studies we identified the first targets of a SoxB1 protein during primary neurogenesis. Sox3 functions as an activator to induce expression of the early neural genes, sox2 and geminin in the absence of protein synthesis and to indirectly inhibit the Bmp target Xvent2. As a result, Sox3 increases cell proliferation, delays neurogenesis and inhibits epidermal and neural crest formation to expand the neural plate. Our studies indicate that Sox3 and 2 have many similar functions in this process including the ability to activate expression of geminin in naïve ectodermal explants. However, there are some differences; Sox3 activates the expression of sox2, while Sox2 does not activate expression of sox3 and sox3 is uniquely expressed throughout the ectoderm prior to neural induction suggesting a role in neural competence. With morpholino-mediated knockdown of Sox3, we demonstrate that it is required for induction of neural tissue by BMP inhibition. Together these data indicate that Sox3 has multiple roles in early neural development including as a factor required for nogginmediated neural induction.
Assuntos
Ectoderma/metabolismo , Neurônios/metabolismo , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Animais , Biomarcadores/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Diferenciação Celular , Proliferação de Células , Ectoderma/embriologia , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Neurônios/citologia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Células-Tronco/citologia , Fatores de Transcrição/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/genéticaRESUMO
DNA replication in Xenopus laevis is extremely reliable, failing to complete before cell division no more than once in 10 000 times; yet replication origin sites are located and initiated stochastically. Using a model based on 1D theories of nucleation and growth and using concepts from extreme-value statistics, we derive the distribution of replication times given a particular initiation function. We show that the experimentally observed initiation strategy for Xenopus laevis meets the reliability constraint and is close to the one that requires the fewest resources of a cell.
Assuntos
Replicação do DNA , Modelos Genéticos , Origem de Replicação , Processos Estocásticos , Xenopus laevis/genética , Animais , Ciclo Celular , Simulação por Computador , Método de Monte CarloRESUMO
Estrogenic endocrine-disrupting chemicals abnormally stimulate vitellogenin gene expression and production in the liver of many male aquatic vertebrates. However, very few studies demonstrate the effects of estrogenic pollutants on brain function. We have used polyethylenimine-mediated in vivo somatic gene transfer to introduce an estrogen response element-thymidine kinase-luciferase (ERE-TK-LUC) construct into the brain. To determine if waterborne estrogenic chemicals modulate gene transcription in the brain, we injected the estrogen-sensitive construct into the brains of Nieuwkoop-Faber stage 54 Xenopus laevis tadpoles. Both ethinylestradiol (EE2; p < 0.002) and bisphenol A (BPA; p < 0.03) increased luciferase activity by 1.9- and 1.5-fold, respectively. In contrast, low physiologic levels of 17ss-estradiol had no effect (p > 0.05). The mixed antagonist/agonist tamoxifen was estrogenic in vivo and increased (p < 0.003) luciferase activity in the tadpole brain by 2.3-fold. There have been no previous reports of somatic gene transfer to the fish brain; therefore, it was necessary to optimize injection and transfection conditions for the adult goldfish (Carassius auratus). Following third brain ventricle injection of cytomegalovirus (CMV)-green fluorescent protein or CMV-LUC gene constructs, we established that cells in the telencephalon and optic tectum are transfected. Optimal transfections were achieved with 1 microg DNA complexed with 18 nmol 22 kDa polyethylenimine 4 days after brain injections. Exposure to EE2 increased brain luciferase activity by 2-fold in males (p < 0.05) but not in females. Activation of an ERE-dependent luciferase reporter gene in both tadpole and fish indicates that waterborne estrogens can directly modulate transcription of estrogen-responsive genes in the brain. We provide a method adaptable to aquatic organisms to study the direct regulation of estrogen-responsive genes in vivo.