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1.
Methods Mol Biol ; 1976: 107-119, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30977069

RESUMO

Mammalian development occurs in utero, which makes it difficult to study the diverse morphogenetic events of neural crest cell development in vivo. Analyses of fixed samples in conjunction with histological methods to evaluate the spatiotemporal roles of specific genes of interest only provide snapshots of mammalian neural crest cell development. This chapter describes methods for isolating and culturing mouse embryos and their organs in vitro, outside the mother, to facilitate real-time imaging and functional analyses of the dynamics of neural crest cell development.


Assuntos
Crista Neural/citologia , Animais , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Eletroporação , Técnicas de Cultura Embrionária , Embrião de Mamíferos/citologia , Camundongos
2.
Macromol Rapid Commun ; 39(4)2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29210526

RESUMO

The spectra of conducting polymers obtained using ultraviolet photoelectron spectroscopy (UPS) exhibit a typical broadening of the tail σUPS ≈ 1 eV, which by an order of magnitude exceeds a commonly accepted value of the broadening of the tail of the density of states σDOS ≈ 0.1 eV obtained using transport measurements. In this work, an origin of this anomalous broadening of the tail of the UPS spectra in a doped conducting polymer, PEDOT (poly(3,4-ethylenedioxythiophene)), is discussed. Based on the semiempirical approach and using a realistic morphological model, the density of valence states in PEDOT doped with molecular counterions is computed. It is shown that due to a disordered character of the material with randomly distributed counterions, the localized charge carriers in PEDOT crystallites experience spatially varying electrostatic potential. This leads to spatially varying local vacuum levels and binding energies. Taking this variation into account the UPS spectrum is obtained with the broadening of the tail comparable to the experimentally observed one. The results imply that the observed broadening of the tail of the UPS spectra in PEDOT provides information about a disordered spatially varying potential in the material rather than the broadening of the DOS itself.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/química , Polímeros/química , Espectroscopia Fotoeletrônica , Eletricidade Estática
3.
Curr Top Dev Biol ; 111: 3-26, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25662256

RESUMO

As vertebrates evolved from protochordates, they shifted to a more predatory lifestyle, and radiated and adapted to most niches of the planet. This process was largely facilitated by the generation of novel vertebrate head structures, which were derived from neural crest cells (NCC). The neural crest is a unique vertebrate cell population that is frequently termed the "fourth germ layer" because it forms in conjunction with the other germ layers and contributes to a diverse array of cell types and tissues including the craniofacial skeleton, the peripheral nervous system, and pigment cells among many other tissues and cell types. NCC are defined by their origin at the neural plate border, via an epithelial-to-mesenchymal transition (EMT), together with multipotency and polarized patterns of migration. These defining characteristics, which evolved independently in the germ layers of invertebrates, were subsequently co-opted through their gene regulatory networks to form NCC in vertebrates. Moreover, recent data suggest that the ability to undergo an EMT was one of the latter features co-opted by NCC. In this review, we discuss the potential origins of NCC and how they evolved to contribute to nearly all tissues and organs throughout the body, based on paleontological evidence together with an evaluation of the evolution of molecules involved in NCC development and their migratory cell paths.


Assuntos
Evolução Biológica , Movimento Celular/fisiologia , Transição Epitelial-Mesenquimal/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Redes Reguladoras de Genes/fisiologia , Crista Neural/citologia , Vertebrados/embriologia , Animais , Regulação da Expressão Gênica no Desenvolvimento/genética , Redes Reguladoras de Genes/genética
4.
PLoS One ; 9(1): e86784, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24475179

RESUMO

Members of the plakophilin-catenin sub-family (Pkp-1, -2, and -3) facilitate the linkage of desmosome junctional components to each other (e.g. desmosomal cadherins to desmoplakin) and the intermediate-filament cytoskeleton. Pkps also contribute to desmosomal stabilization and the trafficking of its components. The functions of Pkps outside of the desmosome are less well studied, despite evidence suggesting their roles in mRNA regulation, small-GTPase modulation (e.g. mid-body scission) during cell division, and cell survival following DNA damage. Pkp-catenins are further believed to have roles in the nucleus given their nuclear localization in some contexts and the known nuclear roles of structurally related catenins, such as beta-catenin and p120-catenin. Further, Pkp-catenin activities in the nuclear compartment have become of increased interest with the identification of interactions between Pkp2-catenin and RNA Pol III and Pkp1 with single-stranded DNA. Consistent with earlier reports suggesting possible nuclear roles in development, we previously demonstrated prominent nuclear localization of Pkp3 in Xenopus naïve ectoderm ("animal cap") cells and recently resolved a similar localization in mouse embryonic stem cells. Here, we report the association and positive functional interaction of Pkp3 with a transcription factor, Ets variant gene 1 (ETV1), which has critical roles in neural development and prominent roles in human genetic disease. Our results are the first to report the interaction of a sequence-specific transcription factor with any Pkp. Using Xenopus laevis embryos and mammalian cells, we provide evidence for the Pkp3:ETV1 complex on both biochemical and functional levels.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/genética , Placofilinas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Primers do DNA/genética , Proteínas de Ligação a DNA/genética , Embrião não Mamífero/metabolismo , Imunofluorescência , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Imunoprecipitação , Hibridização In Situ , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido , Xenopus laevis
5.
Prog Mol Biol Transl Sci ; 116: 387-407, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23481204

RESUMO

The prevailing view of canonical Wnt signaling emphasizes the role of beta-catenin acting downstream of Wnt activation to regulate transcriptional activity. However, emerging evidence indicates that other armadillo catenins in vertebrates, such as members of the p120 subfamily, convey parallel signals to the nucleus downstream of canonical Wnt pathway activation. Their study is thus needed to appreciate the networked mechanisms of canonical Wnt pathway transduction, especially as they may assist in generating the diversity of Wnt effects observed in development and disease. In this chapter, we outline evidence of direct canonical Wnt effects on p120 subfamily members in vertebrates and speculate upon these catenins' roles in conjunction with or aside from beta-catenin.


Assuntos
Tatus/metabolismo , Cateninas/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Animais , Transdução de Sinais
6.
PLoS One ; 7(4): e34342, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22496792

RESUMO

The p120-catenin family has undergone a significant expansion during the evolution of vertebrates, resulting in varied functions that have yet to be discerned or fully characterized. Likewise, members of the plakophilins, a related catenin subfamily, are found throughout the cell with little known about their functions outside the desmosomal plaque. While the plakophilin-3 (Pkp3) knockout mouse resulted in skin defects, we find larger, including lethal effects following its depletion in Xenopus. Pkp3, unlike some other characterized catenins in amphibians, does not have significant maternal deposits of mRNA. However, during embryogenesis, two Pkp3 protein products whose temporal expression is partially complimentary become expressed. Only the smaller of these products is found in adult Xenopus tissues, with an expression pattern exhibiting distinctions as well as overlaps with those observed in mammalian studies. We determined that Xenopus Pkp3 depletion causes a skin fragility phenotype in keeping with the mouse knockout, but more novel, Xenopus tailbud embryos are hyposensitive to touch even in embryos lacking outward discernable phenotypes, and we additionally resolved disruptions in certain peripheral neural structures, altered establishment and migration of neural crest, and defects in ectodermal multiciliated cells. The use of two distinct morpholinos, as well as rescue approaches, indicated the specificity of these effects. Our results point to the requirement of Pkp3 in amphibian embryogenesis, with functional roles in a number of tissue types.


Assuntos
Ectoderma/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Crista Neural/metabolismo , Placofilinas/fisiologia , Xenopus laevis/embriologia , Sequência de Aminoácidos , Animais , Western Blotting , Caderinas/genética , Caderinas/metabolismo , Cateninas/genética , Cateninas/metabolismo , Movimento Celular , Desmossomos/metabolismo , Ectoderma/citologia , Feminino , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , Crista Neural/citologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Homologia de Sequência de Aminoácidos , Xenopus laevis/metabolismo
7.
J Cell Sci ; 125(Pt 3): 561-9, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22389395

RESUMO

The Wnt pathways contribute to many processes in cancer and development, with ß-catenin being a key canonical component. p120-catenin, which is structurally similar to ß-catenin, regulates the expression of certain Wnt target genes, relieving repression conferred by the POZ- and zinc-finger-domain-containing transcription factor Kaiso. We have identified the kinase Dyrk1A as a component of the p120-catenin-Kaiso trajectory of the Wnt pathway. Using rescue and other approaches in Xenopus laevis embryos and mammalian cells, we found that Dyrk1A positively and selectively modulates p120-catenin protein levels, thus having an impact on p120-catenin and Kaiso (and canonical Wnt) gene targets such as siamois and wnt11. The Dyrk1A gene resides within the Down's syndrome critical region, which is amplified in Down's syndrome. A consensus Dyrk phosphorylation site in p120-catenin was identified, with a mutant mimicking phosphorylation exhibiting the predicted enhanced capacity to promote endogenous Wnt-11 and Siamois expression, and gastrulation defects. In summary, we report the biochemical and functional relationship of Dyrk1A with the p120-catenin-Kaiso signaling trajectory, with a linkage to canonical Wnt target genes. Conceivably, this work might also prove relevant to understanding the contribution of Dyrk1A dosage imbalance in Down's syndrome.


Assuntos
Cateninas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Via de Sinalização Wnt/fisiologia , Proteínas de Xenopus/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Cateninas/genética , Primers do DNA/genética , Síndrome de Down/genética , Síndrome de Down/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Dados de Sequência Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , RNA Interferente Pequeno/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Transfecção , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Xenopus laevis/genética , Xenopus laevis/metabolismo , delta Catenina
8.
Dev Dyn ; 240(12): 2601-12, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22028074

RESUMO

The novel adaptor protein Kazrin associates with multifunctional entities including p120-subfamily members (ARVCF-, delta-, and p0071-catenin). Critical contributions of Kazrin to development or homeostasis are indicated with respect to ectoderm formation, integrity and keratinocyte differentiation, whereas its presence in varied tissues suggests broader roles. We find that Kazrin is maternally loaded, is expressed across development and becomes enriched in the forming head. Kazrin's potential contributions to craniofacial development were probed by means of knockdown in the prospective anterior neural region. Cartilaginous head structures as well as eyes on injected sides were reduced in size, with molecular markers suggesting an impact upon neural crest cell establishment and migration. Similar effects followed the depletion of ARVCF (or delta-catenin), with Kazrin:ARVCF functional interplay supported upon ARVCF partial rescue of Kazrin knockdown phenotypes. Thus, Kazrin and its associating ARVCF- and delta-catenins, are required to form craniofacial tissues originating from cranial neural crest and precordal plate.


Assuntos
Proteínas do Domínio Armadillo/metabolismo , Cateninas/metabolismo , Moléculas de Adesão Celular/metabolismo , Movimento Celular/fisiologia , Proteínas de Membrana/metabolismo , Crista Neural/embriologia , Organogênese/fisiologia , Fosfoproteínas/metabolismo , Crânio/embriologia , Proteínas de Xenopus/metabolismo , Animais , Proteínas do Domínio Armadillo/genética , Cartilagem/embriologia , Cateninas/genética , Moléculas de Adesão Celular/genética , Olho/embriologia , Técnicas de Silenciamento de Genes , Proteínas de Membrana/genética , Fosfoproteínas/genética , Crânio/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis , delta Catenina
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