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1.
Nat Commun ; 10(1): 1673, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30975984

RESUMO

Accurate chromosome segregation relies on microtubule end conversion, the ill-understood ability of kinetochores to transit from lateral microtubule attachment to durable association with dynamic microtubule plus-ends. The molecular requirements for this conversion and the underlying biophysical mechanisms are elusive. We reconstituted end conversion in vitro using two kinetochore components: the plus end-directed kinesin CENP-E and microtubule-binding Ndc80 complex, combined on the surface of a microbead. The primary role of CENP-E is to ensure close proximity between Ndc80 complexes and the microtubule plus-end, whereas Ndc80 complexes provide lasting microtubule association by diffusing on the microtubule wall near its tip. Together, these proteins mediate robust plus-end coupling during several rounds of microtubule dynamics, in the absence of any specialized tip-binding or regulatory proteins. Using a Brownian dynamics model, we show that end conversion is an emergent property of multimolecular ensembles of microtubule wall-binding proteins with finely tuned force-dependent motility characteristics.


Assuntos
Segregação de Cromossomos , Cinesina/metabolismo , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Animais , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/isolamento & purificação , Proteínas Cromossômicas não Histona/metabolismo , Microscopia de Fluorescência , Modelos Biológicos , Dinâmica não Linear , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Sf9 , Imagem Individual de Molécula , Processos Estocásticos , Proteínas de Xenopus/genética , Proteínas de Xenopus/isolamento & purificação , Proteínas de Xenopus/metabolismo
2.
Mol Cell ; 73(5): 915-929.e6, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30849395

RESUMO

DNA replication errors generate complex chromosomal rearrangements and thereby contribute to tumorigenesis and other human diseases. One mechanism that triggers these errors is mitotic entry before the completion of DNA replication. To address how mitosis might affect DNA replication, we used Xenopus egg extracts. When mitotic CDK (Cyclin B1-CDK1) is used to drive interphase egg extracts into a mitotic state, the replicative CMG (CDC45/MCM2-7/GINS) helicase undergoes ubiquitylation on its MCM7 subunit, dependent on the E3 ubiquitin ligase TRAIP. Whether replisomes have stalled or undergone termination, CMG ubiquitylation is followed by its extraction from chromatin by the CDC48/p97 ATPase. TRAIP-dependent CMG unloading during mitosis is also seen in C. elegans early embryos. At stalled forks, CMG removal results in fork breakage and end joining events involving deletions and templated insertions. Our results identify a mitotic pathway of global replisome disassembly that can trigger replication fork collapse and DNA rearrangements.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclina B1/metabolismo , Dano ao DNA , Replicação do DNA , DNA/biossíntese , Rearranjo Gênico , Mitose , Proteínas Quinases/metabolismo , Proteínas de Xenopus/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/genética , Ciclina B1/genética , DNA/genética , Reparo do DNA , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Manutenção de Minicromossomo/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/metabolismo
3.
Nat Commun ; 10(1): 1083, 2019 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-30842454

RESUMO

VEGFA signaling controls physiological and pathological angiogenesis and hematopoiesis. Although many context-dependent signaling pathways downstream of VEGFA have been uncovered, vegfa transcriptional regulation in vivo remains unclear. Here, we show that the ETS transcription factor, Etv6, positively regulates vegfa expression during Xenopus blood stem cell development through multiple transcriptional inputs. In agreement with its established repressive functions, Etv6 directly inhibits expression of the repressor foxo3, to prevent Foxo3 from binding to and repressing the vegfa promoter. Etv6 also directly activates expression of the activator klf4; reflecting a genome-wide paucity in ETS-binding motifs in Etv6 genomic targets, Klf4 then recruits Etv6 to the vegfa promoter to activate its expression. These two mechanisms (double negative gate and feed-forward loop) are classic features of gene regulatory networks specifying cell fates. Thus, Etv6's dual function, as a transcriptional repressor and activator, controls a major signaling pathway involved in endothelial and blood development in vivo.


Assuntos
Proteína Forkhead Box O3/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteínas Repressoras/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/fisiologia , Animais , Embrião não Mamífero , Endotélio/embriologia , Endotélio/metabolismo , Proteína Forkhead Box O3/antagonistas & inibidores , Proteína Forkhead Box O3/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Redes Reguladoras de Genes/fisiologia , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , Fatores de Transcrição Kruppel-Like/genética , Morfolinos/genética , Oligonucleotídeos Antissenso/genética , Proteínas Proto-Oncogênicas c-ets/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Transdução de Sinais/fisiologia , Somitos/embriologia , Somitos/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas de Xenopus/antagonistas & inibidores , Proteínas de Xenopus/genética
4.
Mol Vis ; 25: 165-173, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30820152

RESUMO

Purpose: The evolutionarily conserved retinal homeobox (Rax) transcription factor is essential for normal eye development in all vertebrates. Despite Rax's biologic significance, the molecular mechanisms underlying Rax molecular function as a transcriptional regulator are poorly defined. The rax gene encodes a conserved octapeptide motif (OP) near the N-terminus and several conserved regions in the C-terminus of unknown function, including the orthopedia, aristaless, rax (OAR) domain and the RX domain. The purpose of this study is to investigate the contribution of these conserved domains in Rax function. Methods: N-and C-terminal deletion and point mutations were generated in Xenopus laevis rax.L (previously known as Rx1A) using PCR-based methods. We examined the ability of mutated Rax to transactivate a reporter gene consisting of a portion of a rax target gene promoter (from the Xenopus rhodopsin gene) fused to a firefly luciferase coding region and transfected into human embryonic kidney 293T (HEK293T) cells. Portions of the Rax C-terminal region were also assayed for transactivation activity in the context of a heterologous DNA binding domain with an appropriate reporter gene. Results: Full-length Rax weakly activated the reporter. Deletion of the Rax C-terminus increased Rax activity, suggesting that the C-terminus functions to repress Rax activity. Further deletion eventually resulted in a decrease in activity, suggesting that the C-terminal region also can function to enhance Rax activity. Deletion or mutation of the OP motif resulted in a slight decrease in Rax activity. Mutation or deletion of the N-terminal OP motif resulted in a mild decrease in activity and dampened the activity levels of the C-terminal deletions. Further, fusion of the C-terminus of Rax to a heterologous DNA binding domain enhanced transactivation. Conclusions: The present data indicate that the C-terminus of Rax can function to repress or activate transcription in a context-dependent manner. These data support our hypothesis that the highly conserved OAR domain, in combination with other regulatory elements in the Rax C-terminus, coordinates Rax activity, perhaps through functional interaction with the N-terminal OP motif. Taken together, these data provide insight into the structural features that regulate Rax activity.


Assuntos
Sequência de Bases , Proteínas do Olho/genética , Proteínas Recombinantes de Fusão/genética , Retina/metabolismo , Deleção de Sequência , Ativação Transcricional , Proteínas de Xenopus/genética , Motivos de Aminoácidos , Animais , Proteínas do Olho/química , Proteínas do Olho/metabolismo , Expressão Gênica , Genes Reporter , Células HEK293 , Humanos , Luciferases/genética , Luciferases/metabolismo , Mutação Puntual , Regiões Promotoras Genéticas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Xenopus laevis
5.
Chem Biol Interact ; 304: 168-172, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30894314

RESUMO

ALDH16 is a novel family of the aldehyde dehydrogenase (ALDH) superfamily with unique structural characteristics that distinguish it from the other ALDH superfamily members. In addition to structural characteristics, there is an evolutionary-related grouping within the ALDH 16 genes. The ALDH16 isozymes in frog, lower animals, and bacteria possess a critical Cys residue in their active site, which is absent from ALDH16 in mammals and fish. Genomic analysis and plasma metabolomic studies have associated ALDH16A1 with the pathogenesis of gout in humans, although its actual involvement in this disease is poorly understood. Insight into the structure of ALDH16A1 is an important step in deciphering its function in gout. Herein, we report our efforts towards the structural characterization of Xenopus tropicalis ALDH16B1 (the homolog of human ALDH16A1) that was predicted to be catalytically-active. Recombinant ALDH16B1 was expressed in Sf9 cells and purified using affinity and size exclusion chromatography. Crystallization of ALDH16B1 was achieved by vapor diffusion. A data set was collected at 2.5 Šand preliminary crystallographic analysis showed that the frog ALDH16B1 crystals belong to the P 212 121 space group with unit cell parameters a = 80.48 Å, b = 89.73 Å, c = 190.92 Å, α = ß = γ = 90.00°. Structure determination is currently in progress.


Assuntos
Proteínas de Xenopus , Xenopus , Aldeído Desidrogenase/química , Aldeído Desidrogenase/metabolismo , Animais , Biocatálise , Cromatografia em Gel , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Proteínas de Xenopus/isolamento & purificação
6.
Genesis ; 57(5): e23293, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30912273

RESUMO

The Agr family genes, Ag1, Agr2, and Agr3, encode for the thioredoxin domain containing secreted proteins and are specific only for vertebrates. These proteins are attracting increasing attention due to their involvement in many physiological and pathological processes, including exocrine secretion, cancer, regeneration of the body appendages, and the early brain development. At the same time, the mode by which Agrs regulate intracellular processes are poorly understood. Despite that the receptor to Agr2, the membrane anchored protein Prod1, has been firstly discovered in Urodeles, and it has been shown to interact with Agr2 in the regenerating limb, no functional homologs of Prod1 were identified in other vertebrates. This raises the question of the mechanisms by which Agrs can regulate regeneration in other lower vertebrates. Recently, we have identified that Tfp4 (three-fingers Protein 4), the structural and functional homolog of Prod1 in Anurans, interacts with Agr2 in Xenopus laevis embryos. In the present work we show by several methods that the activity of Tfp4 is essential for the tadpole tail regeneration as well as for the early eye and forebrain development during embryogenesis. These data show for the first time the common molecular mechanism of regeneration regulation in amphibians by interaction of Prod1 and Agr2 proteins.


Assuntos
Arginase/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Regeneração/fisiologia , Proteínas de Xenopus/metabolismo , Animais , Proteínas de Transporte/metabolismo , Desenvolvimento Embrionário , Extremidades/embriologia , Larva/genética , Larva/metabolismo , Organogênese , Ligação Proteica/fisiologia , Regeneração/genética , Tiorredoxinas/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/metabolismo
7.
Gene Expr Patterns ; 32: 18-27, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30844509

RESUMO

Guanine nucleotide exchange factors (GEFs) activate Rho GTPases by accelerating their GDP/GTP exchange. Trio and its paralog Kalirin (Kalrn) are unique members of the Rho-GEFs that harbor three catalytic domains: two functional GEF domains and a serine/threonine kinase domain. The N-terminal GEF domain activates Rac1 and RhoG GTPases, while the C-terminal GEF domain acts specifically on RhoA. Trio and Kalrn have an evolutionary conserved function in morphogenetic processes including neuronal development. De novo mutations in TRIO have lately been identified in patients with intellectual disability, suggesting that this protein family plays an important role in development and disease. Phylogenetic and domain analysis revealed that a Kalrn/Trio ancestor originated in Prebilateria and duplicated in Urbilateria to yield Kalrn and Trio. Only few taxa outside the vertebrates retained both of these highly conserved proteins. To obtain first insights into their redundant or distinct functions in a vertebrate model system, we show for the first time a detailed comparative analysis of trio and kalrn expression in Xenopus laevis development. The mRNAs are maternally transcribed and expression increases starting with neurula stages. Trio and kalrn are detected in mesoderm/somites and different neuronal populations in the neural plate/tube and later also in the brain. However, only trio is expressed in migrating neural crest cells, while kalrn expression is detected in the cranial nerves, suggesting distinct functions. Thus, our expression analysis provides a good basis for further functional studies.


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Sequência de Aminoácidos , Animais , Evolução Molecular , Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Humanos , Filogenia , Proteínas Serina-Treonina Quinases/fisiologia , Transdução de Sinais , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Proteínas rho de Ligação ao GTP , Proteína rhoA de Ligação ao GTP
8.
Mol Cell ; 73(3): 574-588.e7, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30595436

RESUMO

DNA-protein crosslinks (DPCs) are bulky lesions that interfere with DNA metabolism and therefore threaten genomic integrity. Recent studies implicate the metalloprotease SPRTN in S phase removal of DPCs, but how SPRTN is targeted to DPCs during DNA replication is unknown. Using Xenopus egg extracts that recapitulate replication-coupled DPC proteolysis, we show that DPCs can be degraded by SPRTN or the proteasome, which act as independent DPC proteases. Proteasome recruitment requires DPC polyubiquitylation, which is partially dependent on the ubiquitin ligase activity of TRAIP. In contrast, SPRTN-mediated DPC degradation does not require DPC polyubiquitylation but instead depends on nascent strand extension to within a few nucleotides of the lesion, implying that polymerase stalling at the DPC activates SPRTN on both leading and lagging strand templates. Our results demonstrate that SPRTN and proteasome activities are coupled to DNA replication by distinct mechanisms that promote replication across immovable protein barriers.


Assuntos
Reparo do DNA , Replicação do DNA , DNA/biossíntese , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , DNA/química , DNA/genética , Feminino , Masculino , Conformação de Ácido Nucleico , Complexo de Endopeptidases do Proteassoma/genética , Domínios e Motivos de Interação entre Proteínas , Proteólise , Células Sf9 , Relação Estrutura-Atividade , Ubiquitinação , Proteínas de Xenopus/genética , Xenopus laevis/genética
9.
Dev Biol ; 446(1): 68-79, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30529252

RESUMO

The specialized sensory organs of the vertebrate head are derived from thickened patches of cells in the ectoderm called cranial sensory placodes. The developmental program that generates these placodes and the genes that are expressed during the process have been studied extensively in a number of animals, yet very little is known about how these genes regulate one another. We previously found via a microarray screen that Six1, a known transcriptional regulator of cranial placode fate, up-regulates Irx1 in ectodermal explants. In this study, we investigated the transcriptional relationship between Six1 and Irx1 and found that they reciprocally regulate each other throughout cranial placode and otic vesicle formation. Although Irx1 expression precedes that of Six1 in the neural border zone, its continued and appropriately patterned expression in the pre-placodal region (PPR) and otic vesicle requires Six1. At early PPR stages, Six1 expands the Irx1 domain, but this activity subsides over time and changes to a predominantly repressive effect. Likewise, Irx1 initially expands Six1 expression in the PPR, but later represses it. We also found that Irx1 and Sox11, a known direct target of Six1, reciprocally affect each other. This work demonstrates that the interactions between Six1 and Irx1 are continuous during PPR and placode development and their transcriptional effects on one another change over developmental time.


Assuntos
Orelha Interna/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas do Tecido Nervoso/genética , Placa Neural/metabolismo , Fatores de Transcrição/genética , Proteínas de Xenopus/genética , Animais , Orelha Interna/citologia , Orelha Interna/embriologia , Ectoderma/citologia , Ectoderma/embriologia , Ectoderma/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Cabeça/embriologia , Proteínas de Homeodomínio/metabolismo , Hibridização In Situ , Proteínas do Tecido Nervoso/metabolismo , Placa Neural/citologia , Placa Neural/embriologia , Fatores de Transcrição SOXC/genética , Fatores de Transcrição SOXC/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis
10.
Methods Mol Biol ; 1874: 507-524, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30353533

RESUMO

The anuran Xenopus laevis has been studied for decades as a model for vertebrate cell and developmental biology. More recently, the highly related species Xenopus tropicalis has offered the opportunity to carry out genetic studies due to its diploid genome as compared to the pseudo-tetraploid Xenopus laevis. Amphibians undergo a biphasic development: embryogenesis to produce a free-living tadpoles and subsequent metamorphosis to transform the tadpole to a frog. This second phase mimics the so-called postembryonic development in mammals when many organs/tissues mature into their adult form in the presence of high levels of plasma thyroid hormone (T3). The total dependence of amphibian metamorphosis on T3 offers a unique opportunity to study postembryonic development in vertebrates, especially with the recent development gene editing technologies that function in amphibians. Here, we first review the basic molecular understanding of the regulation of Xenopus metamorphosis by T3 and T3 receptors (TRs), and then describe a detailed method to use CRISPR to knock out the TR-coactivator SRC3 (steroid receptor coactivator 3), a histone acetyltransferase, in order to study its involvement in gene regulation by T3 in vivo and Xenopus development.


Assuntos
Edição de Genes/métodos , Microinjeções/métodos , Receptores dos Hormônios Tireóideos/metabolismo , Tri-Iodotironina/metabolismo , Xenopus/genética , Animais , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Metamorfose Biológica , Ativação Transcricional , Proteínas de Xenopus/genética
11.
Int J Mol Med ; 43(2): 1105-1113, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30569092

RESUMO

Epigenetic modifier lysine demethylase 3a (Kdm3a) specifically demethylates mono­ and di­methylated ninth lysine of histone 3 and belongs to the Jumonji domain­containing group of demethylases. Kdm3a serves roles during various biological and pathophysiological processes, including spermatogenesis and metabolism, determination of sex, androgen receptor­mediated transcription and embryonic carcinoma cell differentiation. In the present study, physiological functions of Kdm3a were evaluated during embryogenesis of Xenopus laevis. Spatiotemporal expression pattern indicated that kdm3a exhibited its expression from early embryonic stages until tadpole stage, however considerable increase of kdm3a expression was observed during the neurula stage of Xenopus development. Depleting kdm3a using kdm3a antisense morpholino oligonucleotides induced anomalies, including head deformities, small­sized eyes and abnormal pigmentation. Whole­mount in situ hybridization results demonstrated that kdm3a knockdown was associated with defects in neural crest migration. Further, quantitative polymerase chain reaction revealed abnormal expression of neural markers in kdm3a morphants. RNA sequencing of kdm3a morphants indicated that kdm3a was implicated in mesoderm formation, cell adhesion and metabolic processes of embryonic development. In conclusion, the results of the present study indicated that Kdm3a may serve a role in neural development during Xenopus embryogenesis and may be targeted for treatment of developmental disorders. Further investigation is required to elucidate the molecular mechanism underlying the regulation of neural development by Kdm3a.


Assuntos
Desenvolvimento Embrionário/genética , Ossos Faciais/embriologia , Histona Desmetilases com o Domínio Jumonji/genética , Neurogênese/genética , Organogênese/genética , Crânio/embriologia , Proteínas de Xenopus/genética , Animais , Feminino , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Masculino , Xenopus laevis
12.
Dev Biol ; 446(2): 159-167, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30579764

RESUMO

Oscillatory flows of actomyosin play a key role in the migration of single cells in culture and in collective cell movements in Drosophila embryos. In vertebrate embryos undergoing convergent extension (CE), the Planar Cell Polarity (PCP) pathway drives the elongation of the body axis and shapes the central nervous system, and mutations of the PCP genes predispose humans to various malformations including neural tube defects. However, the spatiotemporal patterns of oscillatory actomyosin contractions during vertebrate CE and how they are controlled by the PCP signaling remain unknown. Here, we address these outstanding issues using a combination of in vivo imaging and mathematical modeling. We find that effective execution of CE requires alternative oscillations of cortical actomyosin across cell membranes of neighboring cells within an optimal frequency range. Intriguingly, temporal and spatial clustering of the core PCP protein Prickle 2 (Pk2) is correlated to submembranous accumulations of F-actin, and depletion of Pk2 perturbs the oscillation of actomyosin contractions. These findings shed light on the significance of temporal regulation of actomyosin contraction by the PCP pathway during CE, in addition to its well-studied spatial aspects.


Assuntos
Actomiosina/fisiologia , Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Embrião não Mamífero/citologia , Actomiosina/genética , Algoritmos , Animais , Membrana Celular/metabolismo , Movimento Celular/genética , Polaridade Celular/genética , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Modelos Biológicos , Imagem com Lapso de Tempo/métodos , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis
13.
Dev Biol ; 447(2): 200-213, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30578761

RESUMO

Leukemia inhibitory factor (LIF) is a cytokine member of the interleukin 6 family (IL6) of cytokines. It signals through a heterodimer receptor complex that consists of the LIF receptor (or LIFR formerly known as gp190) and the Interleukin 6 signal transducer (or IL6ST formerly known as gp130). LIF signaling is mediated mainly by signal transducer and activator of transcription 3 (STAT3) and has a wide variety of biological activities with pleiotropic effects on many cell types and organs among which are stem cell renewal and implantation process in mammalian embryo. Despite the wealth of data on LIF in mammalian cells, there is a paucity of information on its functions in lower vertebrates. Here, we provide information on the status and the function of LIF signaling in Xenopus amphibian. The IL6 cytokine family is highly conserved in Xenopus genome both at ligands and receptors levels. All cytokines and receptors of the family, except oncostatin M (OSM) and IL27, can be identified in the genome including the orthologs of LIF, cardiotrophin 1 (CTF1), ciliary neurotrophic factor (CNTF), cardiotrophin like cytokine factor 1 (CLCF1), LIFR, IL6ST, IL6R, IL11RA and CNTFR. Lif mRNA is zygotically expressed after midblastula transition while lifr and il6st are maternally expressed. We have investigated the functions of LIF in Xenopus early development with a gain-of-function analysis combined to the use of a dominant negative form of the receptor. The overexpression of Xenopus lif in embryo activates STAT3 phosphorylation and induces a dramatic phenotype where embryos are ventralised and show a reduction of anterior structures with microcephaly. This results mainly from BMP signal stimulation and antagonism towards IGF signals. In addition, most embryos develop tumor-like cell masses according to both autonomous and non-autonomous processes. Through the use of a dominant negative form of the receptor, we demonstrate for the first time that a functional LIF signaling is required for normal vertebrate kidney development. Owing to its experimental advantages, the Xenopus embryo constitutes a useful model to identify the molecular actors that may account for the pleiotropic functions of LIF and their role in vertebrate development.


Assuntos
Embrião não Mamífero/embriologia , Desenvolvimento Embrionário , Mutação com Ganho de Função , Genes Dominantes , Fator Inibidor de Leucemia/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Xenopus/metabolismo , Animais , Embrião não Mamífero/citologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Fator Inibidor de Leucemia/genética , Proteínas de Xenopus/genética , Xenopus laevis
14.
Mol Cells ; 41(12): 1061-1071, 2018 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-30590909

RESUMO

From Xenopus embryo studies, the BMP4/Smad1-targeted gene circuit is a key signaling pathway for specifying the cell fate between the ectoderm and neuro-ectoderm as well as the ventral and dorsal mesoderm. In this context, several BMP4/Smad1 target transcriptional factors have been identified as repressors of the neuro-ectoderm. However, none of these direct target transcription factors in this pathway, including GATA1b, Msx1 and Ventx1.1 have yet been proven as direct repressors of early neuro-ectodermal gene expression. In order to demonstrate that Ventx1.1 is a direct repressor of neuro-ectoderm genes, a genome-wide Xenopus ChIP-Seq of Ventx1.1 was performed. In this study, we demonstrated that Ventx1.1 bound to the Ventx1.1 response cis-acting element 1 and 2 (VRE1 and VRE2) on the promoter for zic3, which is a key early neuro-ectoderm gene, and this Ventx1.1 binding led to repression of zic3 transcription. Site-directed mutagenesis of VRE1 and VRE2 within zic3 promoter completely abolished the repression caused by Ventx1.1. In addition, we found both the positive and negative regulation of zic3 promoter activity by FoxD5b and Xcad2, respectively, and that these occur through the VREs and via modulation of Ventx1.1 levels. Taken together, the results demonstrate that the BMP4/Smad1 target gene, Ventx1.1, is a direct repressor of neuro-ectodermal gene zic3 during early Xenopus embryogenesis.


Assuntos
Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/genética , Animais , Diferenciação Celular , Sistema Nervoso , Transdução de Sinais , Proteínas de Xenopus/metabolismo
15.
Proc Natl Acad Sci U S A ; 115(39): E9135-E9144, 2018 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-30209221

RESUMO

The Xenopus laevis embryo has been subjected to almost saturating screens for molecules specifically expressed in dorsal Spemann organizer tissue. In this study, we performed high-throughput RNA sequencing of ectodermal explants, called animal caps, which normally give rise to epidermis. We analyzed dissociated animal cap cells that, through sustained activation of MAPK, differentiate into neural tissue. We also microinjected mRNAs for Cerberus, Chordin, FGF8, BMP4, Wnt8, and Xnr2, which induce neural or other germ layer differentiations. The searchable database provided here represents a valuable resource for the early vertebrate cell differentiation. These analyses resulted in the identification of a gene present in frog and fish, which we call Bighead. Surprisingly, at gastrula, it was expressed in the Spemann organizer and endoderm, rather than in ectoderm as we expected. Despite the plethora of genes already mined from Spemann organizer tissue, Bighead encodes a secreted protein that proved to be a potent inhibitor of Wnt signaling in a number of embryological and cultured cell signaling assays. Overexpression of Bighead resulted in large head structures very similar to those of the well-known Wnt antagonists Dkk1 and Frzb-1. Knockdown of Bighead with specific antisense morpholinos resulted in embryos with reduced head structures, due to increased Wnt signaling. Bighead protein bound specifically to the Wnt coreceptor lipoprotein receptor-related protein 6 (Lrp6), leading to its removal from the cell surface. Bighead joins two other Wnt antagonists, Dkk1 and Angptl4, which function as Lrp6 endocytosis regulators. These results suggest that endocytosis plays a crucial role in Wnt signaling.


Assuntos
Endocitose/fisiologia , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia , Angiopoietinas/genética , Angiopoietinas/metabolismo , Animais , Endoderma/citologia , Endoderma/metabolismo , Gástrula/citologia , Gástrula/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Ligação Proteica , Proteínas Wnt/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis
16.
Neural Dev ; 13(1): 22, 2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-30219101

RESUMO

BACKGROUND: Proper patterning of dendritic and axonal arbors is a critical step in the formation of functional neuronal circuits. Developing circuits rely on an array of molecular cues to shape arbor morphology, but the underlying mechanisms guiding the structural formation and interconnectivity of pre- and postsynaptic arbors in real time remain unclear. Here we explore how Down syndrome cell adhesion molecule (DSCAM) differentially shapes the dendritic morphology of central neurons and their presynaptic retinal ganglion cell (RGC) axons in the developing vertebrate visual system. METHODS: The cell-autonomous role of DSCAM, in tectal neurons and in RGCs, was examined using targeted single-cell knockdown and overexpression approaches in developing Xenopus laevis tadpoles. Axonal arbors of RGCs and dendritic arbors of tectal neurons were visualized using real-time in vivo confocal microscopy imaging over the course of 3 days. RESULTS: In the Xenopus visual system, DSCAM immunoreactivity is present in RGCs, cells in the optic tectum and the tectal neuropil at the time retinotectal synaptic connections are made. Downregulating DSCAM in tectal neurons significantly increased dendritic growth and branching rates while inducing dendrites to take on tortuous paths. Overexpression of DSCAM, in contrast, reduced dendritic branching and growth rate. Functional deficits mediated by tectal DSCAM knockdown were examined using visually guided behavioral assays in swimming tadpoles, revealing irregular behavioral responses to visual stimulus. Functional deficits in visual behavior also corresponded with changes in VGLUT/VGAT expression, markers of excitatory and inhibitory transmission, in the tectum. Conversely, single-cell DSCAM knockdown in the retina revealed that RGC axon arborization at the target is influenced by DSCAM, where axons grew at a slower rate and remained relatively simple. In the retina, dendritic arbors of RGCs were not affected by the reduction of DSCAM expression. CONCLUSIONS: Together, our observations implicate DSCAM in the control of both pre- and postsynaptic structural and functional connectivity in the developing retinotectal circuit, where it primarily acts as a neuronal brake to limit and guide postsynaptic dendrite growth of tectal neurons while it also facilitates arborization of presynaptic RGC axons cell autonomously.


Assuntos
Moléculas de Adesão Celular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurônios/citologia , Sinapses/metabolismo , Vias Visuais/citologia , Vias Visuais/crescimento & desenvolvimento , Proteínas de Xenopus/metabolismo , Animais , Aprendizagem da Esquiva/fisiologia , Axônios/metabolismo , Moléculas de Adesão Celular/genética , Dendritos/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Processamento de Imagem Assistida por Computador , Microscopia Confocal , Morfolinos/genética , Morfolinos/metabolismo , Morfolinos/farmacologia , Neurônios/metabolismo , Estimulação Luminosa/efeitos adversos , Retina/citologia , Retina/crescimento & desenvolvimento , Colículos Superiores/citologia , Colículos Superiores/crescimento & desenvolvimento , Sinapses/efeitos dos fármacos , Transfecção , Proteínas Vesiculares de Transporte de Glutamato/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis
17.
Dev Biol ; 442(2): 262-275, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30071218

RESUMO

In anamniotes, somite compartimentalization in the lateral somitic domain leads simultaneously to myotome and dermomyotome formation. In the myotome, Xenopus Sox5 is co-expressed with Myod1 in the course of myogenic differentiation. Here, we studied the function of Sox5 using a Myod1-induced myogenic transcription assay in pluripotent cells of animal caps. We found that Sox5 enhances myogenic transcription of muscle markers Des, Actc1, Ckm and MyhE3. The use of chimeric transactivating or transrepressive Sox5 proteins indicates that Sox5 acts as a transrepressor and indirectly stimulates myogenic transcription except for the slow muscle-specific genes Myh7L, Myh7S, Myl2 and Tnnc1. We showed that this role is shared by Sox6, which is structurally similar to Sox5, both belonging to the SoxD subfamily of transcription factors. Moreover, Sox5 can antagonize the inhibitory function of Meox2 on myogenic differentiation. Meox2 which is a dermomyotome marker, represses myogenic transcription in Myod-induced myogenic transcription assay and in Nodal5-induced mesoderm from animal cap assay. The inhibitory function of Meox2 and the pro-myogenic function of Sox5 were confirmed during Xenopus normal development by the use of translation-blocking oligomorpholinos and dexamethasone inducible chimeric Sox5 and Meox2 proteins. We have therefore identified a new function for SoxD proteins in muscle cells, which can indirectly enhance myogenic transcription through transrepression, in addition to the previously identified function as a direct repressor of slow muscle-specific genes.


Assuntos
Fatores de Transcrição SOXD/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Diferenciação Celular/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Mesoderma/metabolismo , Células Musculares/metabolismo , Desenvolvimento Muscular/genética , Músculos/metabolismo , Proteína MyoD/genética , Proteína MyoD/metabolismo , Fatores de Transcrição SOXD/genética , Somitos/metabolismo , Ativação Transcricional/fisiologia , Proteínas de Xenopus/genética , Xenopus laevis
18.
Artigo em Inglês | MEDLINE | ID: mdl-30055282

RESUMO

Phthalates are used worldwide in the manufacturing of plastics, added to cosmetic products, personal care products, pharmaceuticals, medical devices, and paints; and are widely detected in soil, surface water, and organism tissues. Phthalate esters have been previously shown to interfere with the endocrine system in vertebrates. However, few studies have investigated the effects of phthalates on testosterone-converting enzymes that affect hormone levels and reproduction. In the present study, we exposed the Western clawed frog (Silurana tropicalis) to 0.1, 1, and 10 µM diethylhexyl phthalate (DEHP), dibutyl phthalate (DBP), and diethyl phthalate (DEP) during early amphibian embryonic development. Additional DBP exposures were conducted ex vivo using mature frog testes. Malformations and mRNA levels of genes associated to reproduction and oxidative stress were evaluated. 0.1 µM DEHP, DBP, and DEP induced an array of malformations, including incomplete gut coiling, edemas, and eye malformations. Moreover, all three phthalates increased the expression of androgen-related genes, such as steroid-5α-reductase 1, 2, 3, steroid-5ß-reductase, and androgen receptor at concentrations ranging from 0.1 to 10 µM depending on the phthalate and gene. Data suggest that the phthalate esters tested are teratogens to the amphibian embryo and that these phthalates exhibit an androgenic activity in amphibians.


Assuntos
3-Oxo-5-alfa-Esteroide 4-Desidrogenase/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Disruptores Endócrinos/toxicidade , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Ácidos Ftálicos/toxicidade , Xenopus/embriologia , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , Animais , Dibutilftalato/toxicidade , Dietilexilftalato/toxicidade , Embrião não Mamífero/anormalidades , Embrião não Mamífero/metabolismo , Poluentes Ambientais/toxicidade , Isoenzimas/genética , Isoenzimas/metabolismo , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Larva/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Oxirredutases/genética , Oxirredutases/metabolismo , Plastificantes/toxicidade , Reprodutibilidade dos Testes , Teratogênios/toxicidade , Xenopus/anormalidades , Xenopus/crescimento & desenvolvimento , Xenopus/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo
19.
Elife ; 72018 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-30035713

RESUMO

Neural crest progenitors are specified through the modulation of several signaling pathways, among which the activation of Wnt/ß-catenin signaling by Wnt8 is especially critical. Glycoproteins of the Dickkopf (Dkk) family are important modulators of Wnt signaling acting primarily as Wnt antagonists. Here we report that Dkk2 is required for neural crest specification functioning as a positive regulator of Wnt/ß-catenin signaling. Dkk2 depletion in Xenopus embryos causes a loss of neural crest progenitors, a phenotype that is rescued by expression of Lrp6 or ß-catenin. Dkk2 overexpression expands the neural crest territory in a pattern reminiscent of Wnt8, Lrp6 and ß-catenin gain-of-function phenotypes. Mechanistically, we show that Dkk2 mediates its neural crest-inducing activity through Lrp6 and ß-catenin, however unlike Wnt8, in a GSK3ß independent manner. These findings suggest that Wnt8 and Dkk2 converge on ß-catenin using distinct transduction pathways both independently required to activate Wnt/ß-catenin signaling and induce neural crest cells.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Glicogênio Sintase Quinase 3 beta/metabolismo , Crista Neural/fisiologia , Proteínas de Xenopus/metabolismo , beta Catenina/metabolismo , Animais , Linhagem da Célula , Células Cultivadas , Embrião não Mamífero/citologia , Embrião não Mamífero/fisiologia , Glicogênio Sintase Quinase 3 beta/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Crista Neural/citologia , Via de Sinalização Wnt , Proteínas de Xenopus/genética , Xenopus laevis , beta Catenina/genética
20.
Gen Comp Endocrinol ; 267: 172-182, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-29990494

RESUMO

A series of ex vivo exposures using testicular and ovarian tissues of sexually mature Western clawed frogs (Silurana tropicalis) were designed to examine molecular mechanisms of thyroid hormone (TH) and androgen crosstalk sans hypophyseal feedback as well as investigate potential sex-specific differences. Tissues were exposed ex vivo to either triiodothyronine (T3), iopanoic acid (IOP), one co-treatment of IOP + 5α-dihydrotestosterone (5α-DHT), 5α-DHT, 5ß-dihydrotestosterone (5ß-DHT), or testosterone (T). Direct exposure to different androgens led to androgen specific increases in thyroid receptor and deiodinase transcripts in testes (trß and dio1) but a decrease in expression in ovaries (trß and dio3), suggesting that male and female frogs can be differently affected by androgenic compounds. Moreover, exposure to select androgens differentially increased estrogen-related transcription (estrogen receptor alpha (erα) and aromatase (cyp19)) and production (estradiol) in ovaries and testes indicating the activation of alternate metabolic pathways yielding estrogenic metabolites. Sex-steroid-related transcription (i.e., steroid 5α-reductase type 2 (srd5α2) and erα) and production (i.e., 5α-DHT) were also differentially regulated by THs. The presence and frequency of transcription factor binding sites in the putative promoter regions of TH- and sex steroid-related genes were also examined in S. tropicalis, rodent, and fish models using in silico analysis. In summary, this study provides an improved mechanistic understanding of TH- and androgen-mediated actions and reveals differential transcriptional effects as a function of sex in frogs.


Assuntos
Androgênios/farmacologia , Regulação da Expressão Gênica , Ovário/metabolismo , Maturidade Sexual , Testículo/metabolismo , Hormônios Tireóideos/farmacologia , Xenopus/genética , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/metabolismo , Animais , Di-Hidrotestosterona/metabolismo , Estrogênios/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Ovário/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Maturidade Sexual/efeitos dos fármacos , Testículo/efeitos dos fármacos , Tri-Iodotironina/farmacologia , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo
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