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1.
Acta Neuropathol ; 135(2): 267-283, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29149419

RESUMO

Although a growing body of evidence indicates that phenotypic plasticity exhibited by glioblastoma cells plays a central role in tumor development and post-therapy recurrence, the master drivers of their aggressiveness remain elusive. Here we mapped the changes in active (H3K4me3) and repressive (H3K27me3) histone modifications accompanying the repression of glioblastoma stem-like cells tumorigenicity. Genes with changing histone marks delineated a network of transcription factors related to cancerous behavior, stem state, and neural development, highlighting a previously unsuspected association between repression of ARNT2 and loss of cell tumorigenicity. Immunohistochemistry confirmed ARNT2 expression in cell sub-populations within proliferative zones of patients' glioblastoma. Decreased ARNT2 expression was consistently observed in non-tumorigenic glioblastoma cells, compared to tumorigenic cells. Moreover, ARNT2 expression correlated with a tumorigenic molecular signature at both the tissue level within the tumor core and at the single cell level in the patients' tumors. We found that ARNT2 knockdown decreased the expression of SOX9, POU3F2 and OLIG2, transcription factors implicated in glioblastoma cell tumorigenicity, and repressed glioblastoma stem-like cell tumorigenic properties in vivo. Our results reveal ARNT2 as a pivotal component of the glioblastoma cell tumorigenic signature, located at a node of a transcription factor network controlling glioblastoma cell aggressiveness.


Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias Encefálicas/metabolismo , Cromatina/metabolismo , Glioblastoma/metabolismo , Idoso , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Células Cultivadas , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glioblastoma/genética , Glioblastoma/patologia , Código das Histonas , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Invasividade Neoplásica/fisiopatologia , Transplante de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fator de Transcrição 2 de Oligodendrócitos/metabolismo , Fatores do Domínio POU/metabolismo , Fatores de Transcrição SOX9/metabolismo
2.
Acta Neuropathol ; 133(4): 645-660, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28032215

RESUMO

Cell populations with differing proliferative, stem-like and tumorigenic states co-exist in most tumors and especially malignant gliomas. Whether metabolic variations can drive this heterogeneity by controlling dynamic changes in cell states is unknown. Metabolite profiling of human adult glioblastoma stem-like cells upon loss of their tumorigenicity revealed a switch in the catabolism of the GABA neurotransmitter toward enhanced production and secretion of its by-product GHB (4-hydroxybutyrate). This switch was driven by succinic semialdehyde dehydrogenase (SSADH) downregulation. Enhancing GHB levels via SSADH downregulation or GHB supplementation triggered cell conversion into a less aggressive phenotypic state. GHB affected adult glioblastoma cells with varying molecular profiles, along with cells from pediatric pontine gliomas. In all cell types, GHB acted by inhibiting α-ketoglutarate-dependent Ten-eleven Translocations (TET) activity, resulting in decreased levels of the 5-hydroxymethylcytosine epigenetic mark. In patients, low SSADH expression was correlated with high GHB/α-ketoglutarate ratios, and distinguished weakly proliferative/differentiated glioblastoma territories from proliferative/non-differentiated territories. Our findings support an active participation of metabolic variations in the genesis of tumor heterogeneity.


Assuntos
Neoplasias Encefálicas/metabolismo , Carcinogênese/metabolismo , Glioma/metabolismo , Hidroxibutiratos/metabolismo , Células-Tronco Neoplásicas/metabolismo , Ácido gama-Aminobutírico/metabolismo , Idoso , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/cirurgia , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/cirurgia , Carcinogênese/patologia , Morte Celular/fisiologia , Proliferação de Células/fisiologia , Criança , Pré-Escolar , Feminino , Glioma/patologia , Glioma/cirurgia , Humanos , Masculino , Camundongos Nus , Pessoa de Meia-Idade , Transplante de Neoplasias , Células-Tronco Neoplásicas/patologia , Succinato-Semialdeído Desidrogenase/metabolismo
3.
Biochim Biophys Acta ; 1849(2): 112-21, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24980696

RESUMO

The essential roles of thyroid hormone (TH) in perinatal brain development have been known for decades. More recently, many of the molecular mechanisms underlying the multiple effects of TH on proliferation, differentiation, migration, synaptogenesis and myelination in the developing nervous system have been elucidated. At the same time data from both epidemiological studies and animal models have revealed that the influence of thyroid signaling on development of the nervous system, extends to all periods of life, from early embryogenesis to neurogenesis in the adult brain. This review focuses on recent insights into the actions of TH during early neurogenesis. A key concept is that, in contrast to the previous ideas that only the unliganded receptor was implicated in these early phases, a critical role of the ligand, T3, is increasingly recognized. These findings are considered in the light of increasing knowledge of cell specific control of T3 availability as a function of deiodinase activity and transporter expression. These requirements for TH in the early stages of neurogenesis take on new relevance given the increasing epidemiological data on adverse effects of TH lack in early pregnancy on children's neurodevelopmental outcome. These ideas lead logically into a discussion on how the actions of TH during the first phases of neurogenesis can be potentially disrupted by gestational iodine lack and/or chemical pollution. This article is part of a Special Issue entitled: Nuclear receptors in animal development.


Assuntos
Desenvolvimento Embrionário , Disruptores Endócrinos/toxicidade , Neurogênese , Hormônios Tireóideos/fisiologia , Adulto , Animais , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Feminino , Humanos , Modelos Animais , Sistema Nervoso/efeitos dos fármacos , Sistema Nervoso/metabolismo , Neurogênese/efeitos dos fármacos , Neurogênese/genética , Gravidez , Receptores dos Hormônios Tireóideos/antagonistas & inibidores , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Hormônios Tireóideos/farmacologia
4.
Acta Neuropathol Commun ; 7(1): 155, 2019 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-31619292

RESUMO

Glioblastoma cell ability to adapt their functioning to microenvironment changes is a source of the extensive intra-tumor heterogeneity characteristic of this devastating malignant brain tumor. A systemic view of the metabolic pathways underlying glioblastoma cell functioning states is lacking. We analyzed public single cell RNA-sequencing data from glioblastoma surgical resections, which offer the closest available view of tumor cell heterogeneity as encountered at the time of patients' diagnosis. Unsupervised analyses revealed that information dispersed throughout the cell transcript repertoires encoded the identity of each tumor and masked information related to cell functioning states. Data reduction based on an experimentally-defined signature of transcription factors overcame this hurdle. It allowed cell grouping according to their tumorigenic potential, regardless of their tumor of origin. The approach relevance was validated using independent datasets of glioblastoma cell and tissue transcriptomes, patient-derived cell lines and orthotopic xenografts. Overexpression of genes coding for amino acid and lipid metabolism enzymes involved in anti-oxidative, energetic and cell membrane processes characterized cells with high tumorigenic potential. Modeling of their expression network highlighted the very long chain polyunsaturated fatty acid synthesis pathway at the core of the network. Expression of its most downstream enzymatic component, ELOVL2, was associated with worsened patient survival, and required for cell tumorigenic properties in vivo. Our results demonstrate the power of signature-driven analyses of single cell transcriptomes to obtain an integrated view of metabolic pathways at play within the heterogeneous cell landscape of patient tumors.


Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Aminoácidos/metabolismo , Análise por Conglomerados , Regulação Neoplásica da Expressão Gênica , Humanos , Metabolismo dos Lipídeos , Análise de Célula Única
5.
PLoS One ; 13(4): e0195374, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29641587

RESUMO

Thyroid hormone (TH) orchestrates amphibian metamorphosis. Thus, this developmental phase is often used to study TH-dependent responses in specific tissues. However, TH signaling appears early in development raising the question of the control of TH availability in specific cell types prior to metamorphosis. TH availability is under strict temporal and tissue-specific control by deiodinases. We examined the expression of the TH-inactivating enzyme, deiodinase type 3 (D3), during early retinal development. To this end we created a Xenopus laevis transgenic line expressing GFP from the Xenopus dio3 promoter region (pdio3) and followed pdio3-GFP expression in pre-metamorphic tadpoles. To validate retinal GFP expression in the transgenic line as a function of dio3 promoter activity, we used in situ hybridization to compare endogenous dio3 expression to reporter-driven GFP activity. Retinal expression of dio3 increased during pre-metamorphosis through stages NF41, 45 and 48. Both sets of results show dio3 to have cell-specific, dynamic expression in the pre-metamorphic retina. At stage NF48, dio3 expression co-localised with markers for photoreceptors, rods, Opsin-S cones and bipolar neurons. In contrast, in post-metamorphic juveniles dio3 expression was reduced and spatially confined to certain photoreceptors and amacrine cells. We compared dio3 expression at stages NF41 and NF48 with TH-dependent transcriptional responses using another transgenic reporter line: THbZIP-GFP and by analyzing the expression of T3-regulated genes in distinct TH availability contexts. At stage NF48, the majority of retinal cells expressing dio3 were negative for T3 signaling. Notably, most ganglion cells were virtually both dio3-free and T3-responsive. The results show that dio3 can reduce TH availability at the cellular scale. Further, a reduction in dio3 expression can trigger fine-tuned T3 action in cell-type specific maturation at the right time, as exemplified here in photoreceptor survival in the pre-metamorphic retina.


Assuntos
Regulação Enzimológica da Expressão Gênica , Iodeto Peroxidase/genética , Larva/crescimento & desenvolvimento , Larva/genética , Retina/crescimento & desenvolvimento , Retina/metabolismo , Animais , Metamorfose Biológica , Opsinas/genética , Células Fotorreceptoras Retinianas Cones/metabolismo , Hormônios Tireóideos/metabolismo , Xenopus laevis/genética , Xenopus laevis/crescimento & desenvolvimento
7.
Endocrinology ; 147(7): 3519-29, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16601143

RESUMO

In frogs such as Rana and Xenopus, metamorphosis does not occur in the absence of a functional thyroid gland. Previous studies indicated that coordinated development in frogs requires tissue and stage-dependent type II and type III iodothyronine deiodinase expression patterns to obtain requisite levels of intracellular T(3) in tissues at the appropriate stages of metamorphosis. No type I iodothyronine deiodinase (D1), defined as T(4) or reverse T(3) (rT3) outer-ring deiodinase (ORD) activity with Michaelis constant (K(m)) values in the micromolar range and sensitivity to 6-propyl-2-thiouracil (6-PTU), could be detected in tadpoles so far. We obtained a X. laevis D1 cDNA clone from brain tissue. The complete sequence of this clone (1.1 kb, including poly A tail) encodes an ORF of 252 amino acid residues with high homology to other vertebrate D1 enzymes. The core catalytic center includes a UGA-encoded selenocysteine residue, and the 3' untranslated region (about 300 nt) contains a selenocysteine insertion sequence element. Transfection of cells with an expression vector containing the full-length cDNA resulted in generation of significant deiodinase activity in the homogenates. The enzyme displayed ORD activity with T(4) (K(m) 0.5 microm) and rT3 (K(m) 0.5 microm) and inner-ring deiodinase activity with T(4) (K(m) 0.4 microm). Recombinant Xenopus D1 was essentially insensitive to inhibition by 6-PTU (IC(50) > 1 mm) but was sensitive to gold thioglucose (IC(50) 0.1 mum) and iodoacetate (IC(50) 10 microm). Because the residue 2 positions downstream from the selenocysteine is Pro in Xenopus D1 but Ser in all cloned PTU-sensitive D1 enzymes, we prepared the Pro132Ser mutant of Xenopus D1. The mutant enzyme showed strongly increased ORD activity with T(4) and rT3 (K(m) about 4 microm) and was highly sensitive to 6-PTU (IC(50) 2 microm). Little native D1 activity could be detected in Xenopus liver, kidney, brain, and gut, but significant D1 mRNA expression was observed in juvenile brain and adult liver and kidney. These results indicate the existence of a 6-PTU-insensitive D1 enzyme in X. laevis tissues, but its role during tadpole metamorphosis remains to be defined.


Assuntos
Iodeto Peroxidase/química , Iodeto Peroxidase/genética , Mutação , Prolina/química , Serina/química , Regiões 3' não Traduzidas , Sequência de Aminoácidos , Animais , Sequência de Bases , Domínio Catalítico , Cinética , Dados de Sequência Molecular , Propiltiouracila/farmacologia , Ratos , Selenocisteína/química , Homologia de Sequência de Aminoácidos , Xenopus laevis
8.
Endocrinology ; 147(10): 4941-9, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16825318

RESUMO

Thyroid hormones orchestrate amphibian metamorphosis. The type 2 and type 3 deiodinases make vital contributions to this process by controlling levels of the thyroid hormones T(4) and T(3) available to different tissues. Because the tadpole thyroid gland is not functional until stage NF44, it has been widely assumed that thyroid signaling is absent during amphibian early development, thyroid hormone only becoming a major regulator during premetamorphic stages. Similarly, in mammals, thyroid function is known to be essential to neuronal development, especially during the perinatal stages, but again little is known about early stages of development. Here we demonstrate that key elements of thyroid hormone signaling are present during early development of Xenopus. In particular, we find functional thyroid hormone-activating deiodinases and significant levels of their substrates, T(4) and T(3), during early embryogenesis. Furthermore, we have further characterized a recently identified deiodinase in amphibians, homologous to mammalian type 1 deiodinase (D1). This enzyme is expressed in marked, spatially defined patterns during embryogenesis. The patterns of expression of type 1 deiodinase are distinct from those of type 2 and type 3 deiodinases. Deiodinase expression is found in neurogenic areas from stage NF30 onward, both in the central and peripheral nervous systems. We conclude that both activating and inactivating deiodinases show dynamic patterns of expression during early embryogenesis in amphibians, particularly in neurogenic areas. These findings suggest that thyroid hormone signaling is a key component of early neuronal development in vertebrates.


Assuntos
Embrião não Mamífero/enzimologia , Iodeto Peroxidase/metabolismo , Animais , Desenvolvimento Embrionário , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Hibridização In Situ , Isoenzimas/metabolismo , Metamorfose Biológica , Óvulo/metabolismo , Óvulo/fisiologia , RNA/análise , RNA/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Hormônios Tireóideos/metabolismo , Hormônios Tireóideos/fisiologia , Tiroxina/metabolismo , Tiroxina/fisiologia , Tri-Iodotironina/metabolismo , Xenopus laevis
9.
Int J Dev Biol ; 48(2-3): 217-31, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15272388

RESUMO

The first part of this article is a review of the current status of knowledge of the fish skin, with particular attention to its development. In the second part we present original results obtained in zebrafish (Danio rerio), with particular emphasis on the deposition and organisation of the dermal collagenous stroma. Using a series of zebrafish specimens aged between 15 hours postfertilization (hpf) and 4.5 years old, we have combined Transmission Electron Microscopy (TEM) observations and in situ hybridisation using type I collagen a2 chain (Col1a2) probe. Collagen fibrils, with a diameter of 22 nm, appear first in an acellular subepidermal space at 24 hpf, are first all oriented in the same direction, and form the primary dermal stroma. Subsequently, three events occur. (1) From 5-7 days pf (dpf) onwards the collagen fibrils self-organise into several lamellae arranged in a plywood-like structure, starting in the upper layers and progressing throughout the entire thickness of the dermis. (2) At 20-26 dpf, fibroblasts of unknown origin progressively invade the acellular collagenous stroma, some of them accumulating below the epidermis. (3) Concomitant with the invasion of fibroblasts, the collagen fibrils increase progressively in diameter to reach 160 nm towards the end of the fish life. In situ hybridisation experiments reveal that, between 24 and 48 hpf, the collagen matrix is produced by the epidermis only. From 72 hpf to 20-26 dpf, both the basal epidermal cells and the dermal cells bordering the deep region of the dermis are involved in the production of collagen. When the fibroblasts invade the plywood-like structure, the epidermal cells progressively cease to synthesise collagen, which from this point is produced only by the fibroblasts. This suggests that the fibroblasts secrete a still unidentified signalling molecule that downregulates collagen production by the epidermis.


Assuntos
Colágeno Tipo I/metabolismo , Derme/metabolismo , Pele/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Animais , Movimento Celular , Colágeno/biossíntese , Colágeno/ultraestrutura , Colágeno Tipo I/ultraestrutura , Derme/química , Derme/ultraestrutura , Regulação para Baixo , Embrião não Mamífero/metabolismo , Embrião não Mamífero/ultraestrutura , Epiderme/metabolismo , Epiderme/ultraestrutura , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestrutura , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Fibroblastos/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Larva , Modelos Biológicos , RNA Mensageiro/metabolismo , RNA Mensageiro/ultraestrutura , Pele/embriologia , Pele/ultraestrutura , Fatores de Tempo
10.
Gene Expr Patterns ; 3(3): 351-4, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12799084

RESUMO

Endostatin, located in the NC1 domain of the collagen XVIII, is believed to inhibit the migration and proliferation of endothelial cells (Fed. Am. Soc. Exp. Biol. J. 15 (2001) 1044) and to play a role in axon guidance in Caenorhabditis elegans (J. Cell Biol. 152 (2001) 1219). Zebrafish is an attractive vertebrate model to determine the role of endostatin and the entire molecule of collagen XVIII during vertebrate development. Therefore, we have investigated the expression pattern of COL18A1 in zebrafish embryos from the segmentation to the hatching period stages.


Assuntos
Endostatinas/genética , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Endostatinas/biossíntese , Hibridização In Situ , Dados de Sequência Molecular , Peixe-Zebra/metabolismo
11.
Artigo em Inglês | MEDLINE | ID: mdl-24808891

RESUMO

The vital roles of thyroid hormone in multiple aspects of perinatal brain development have been known for over a century. In the last decades, the molecular mechanisms underlying effects of thyroid hormone on proliferation, differentiation, migration, synaptogenesis, and myelination in the developing nervous system have been gradually dissected. However, recent data reveal that thyroid signaling influences neuronal development throughout life, from early embryogenesis to the neurogenesis in the adult brain. This review deals with the latter phase and analyses current knowledge on the role of T3, the active form of thyroid hormone, and its receptors in regulating neural stem cell function in the hippocampus and the subventricular zone, the two principal sites harboring neurogenesis in the adult mammalian brain. In particular, we discuss the critical roles of T3 and TRα1 in commitment to a neuronal phenotype, a process that entails the repression of a number of genes notably that encoding the pluripotency factor, Sox2. Furthermore, the question of the relevance of thyroid hormone control of adult neurogenesis is considered in the context of brain aging, cognitive decline, and neurodegenerative disease.

12.
PLoS One ; 9(9): e106378, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25184636

RESUMO

BACKGROUND: Inhibitory factors have been implicated in the failure of remyelination in demyelinating diseases. Myelin associated inhibitors act through a common receptor called Nogo receptor (NgR) that plays critical inhibitory roles in CNS plasticity. Here we investigated the effects of abrogating NgR inhibition in a non-immune model of focal demyelination in adult mouse optic chiasm. METHODOLOGY/PRINCIPAL FINDINGS: A focal area of demyelination was induced in adult mouse optic chiasm by microinjection of lysolecithin. To knock down NgR levels, siRNAs against NgR were intracerebroventricularly administered via a permanent cannula over 14 days, Functional changes were monitored by electrophysiological recording of latency of visual evoked potentials (VEPs). Histological analysis was carried out 3, 7 and 14 days post demyelination lesion. To assess the effect of NgR inhibition on precursor cell repopulation, BrdU was administered to the animals prior to the demyelination induction. Inhibition of NgR significantly restored VEPs responses following optic chiasm demyelination. These findings were confirmed histologically by myelin specific staining. siNgR application resulted in a smaller lesion size compared to control. NgR inhibition significantly increased the numbers of BrdU+/Olig2+ progenitor cells in the lesioned area and in the neurogenic zone of the third ventricle. These progenitor cells (Olig2+ or GFAP+) migrated away from this area as a function of time. CONCLUSIONS/SIGNIFICANCE: Our results show that inhibition of NgR facilitate myelin repair in the demyelinated chiasm, with enhanced recruitment of proliferating cells to the lesion site. Thus, antagonizing NgR function could have therapeutic potential for demyelinating disorders such as Multiple Sclerosis.


Assuntos
Doenças Desmielinizantes/terapia , Proteínas da Mielina/antagonistas & inibidores , Bainha de Mielina/metabolismo , Quiasma Óptico/metabolismo , Receptores de Superfície Celular/genética , Recuperação de Função Fisiológica , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Bromodesoxiuridina/administração & dosagem , Movimento Celular , Proliferação de Células , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/patologia , Potenciais Evocados Visuais , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Regulação da Expressão Gênica , Injeções Intraventriculares , Lisofosfatidilcolinas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microinjeções , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , Bainha de Mielina/patologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptor Nogo 1 , Fator de Transcrição 2 de Oligodendrócitos , Quiasma Óptico/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Tempo
13.
Mol Ther Nucleic Acids ; 2: e89, 2013 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-23612115

RESUMO

RNA interference (RNAi) is a major tool for basic and applied investigations. However, obtaining RNAi data that have physiological significance requires investigation of regulations and therapeutic strategies in appropriate in vivo settings. To examine in vivo gene regulation and protein function in the adult neural stem cell (NSC) niche, we optimized a new non-viral vector for delivery of siRNA into the subventricular zone (SVZ). This brain region contains the neural stem and progenitor cells populations that express the stem cell marker, SOX2. Temporally and spatially controlled Sox2 knockdown was achieved using the monocationic lipid vector, IC10. siRNA/IC10 complexes were stable over time and smaller (<40 nm) than jetSi complexes (≈400 nm). Immunocytochemistry showed that siRNA/IC10 complexes efficiently target both the progenitor and stem cell populations in the adult SVZ. Injection of the complexes into the lateral brain ventricle resulted in specific knockdown of Sox2 in the SVZ. Furthermore, IC10-mediated transient in vivo knockdown of Sox2-modulated expression of several genes implicated in NSC maintenance. Taken together, these data show that IC10 cationic lipid formulation can efficiently vectorize siRNA in a specific area of the adult mouse brain, achieving spatially and temporally defined loss of function.Molecular Therapy-Nucleic Acids (2013) 2, e89; doi:10.1038/mtna.2013.8; published online 23 April 2013.

14.
Cell Stem Cell ; 10(5): 531-43, 2012 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-22560077

RESUMO

The subventricular zone (SVZ) neural stem cell niche contains mixed populations of stem cells, transit-amplifying cells, and migrating neuroblasts. Deciphering how endogenous signals, such as hormones, affect the balance between these cell types is essential for understanding the physiology of niche plasticity and homeostasis. We show that Thyroid Hormone (T(3)) and its receptor, TRα1, are directly involved in maintaining this balance. TRα1 is expressed in amplifying and migrating cells. In vivo gain- and loss-of-function experiments demonstrate first, that T(3)/TRα1 directly repress Sox2 expression, and second, that TRα1 overexpression in the niche favors the appearance of DCX+ migrating neuroblasts. Lack of TRα increases numbers of SOX2+ cells in the SVZ. Hypothyroidism increases proportions of cells in interphase. Thus, in the adult SVZ, T(3)/TRα1 together favor neural stem cell commitment and progression toward a migrating neuroblast phenotype; this transition correlates with T(3)/TRα1-dependent transcriptional repression of Sox2.


Assuntos
Células-Tronco Adultas/fisiologia , Células-Tronco Neurais/fisiologia , Neurogênese/genética , Fatores de Transcrição SOXB1/metabolismo , Receptores alfa dos Hormônios Tireóideos/metabolismo , Hormônios Tireóideos/metabolismo , Animais , Movimento Celular/genética , Proteína Duplacortina , Repressão Enzimática/genética , Camundongos , Camundongos Mutantes , RNA Interferente Pequeno/genética , Fatores de Transcrição SOXB1/genética , Transdução de Sinais , Nicho de Células-Tronco/genética , Receptores alfa dos Hormônios Tireóideos/genética , Hormônios Tireóideos/genética , Transgenes/genética
15.
Mol Cell Endocrinol ; 293(1-2): 71-9, 2008 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-18657589

RESUMO

Amphibian metamorphosis is a well-established model for dissecting the mechanisms underlying thyroid hormone (TH) action. How the pro-hormone, T(4), the active form, T(3), the deiodinases and the nuclear receptors (TRs) contribute to metamorphosis in Xenopus has been extensively investigated. Our recent work has concentrated on two key ideas in TH signalling in Xenopus: first, that there could be active roles for both liganded and unliganded receptors, and second, that ligand availability is a determining factor orchestrating these actions and is tightly controlled in target tissues. Recently, we addressed these questions at stages preceding metamorphosis, i.e. during embryogenesis, before differentiation of a functional thyroid gland. We show that repression by unliganded TR is essential to craniofacial and eye development during early development and that at these stages all three deiodinases are active. These results open new perspectives on the potential roles of TH signalling during embryogenesis.


Assuntos
Transdução de Sinais , Hormônios Tireóideos/metabolismo , Animais , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário , Ativação Enzimática , Regulação da Expressão Gênica no Desenvolvimento , Iodeto Peroxidase/química , Iodeto Peroxidase/genética , Iodeto Peroxidase/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Ligantes , Modelos Animais , Receptores dos Hormônios Tireóideos/fisiologia , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis
16.
EMBO J ; 25(20): 4943-51, 2006 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-17006540

RESUMO

Thyroid hormone receptors generally activate transcription of target genes in the presence of thyroid hormone (T(3)) and repress their transcription in its absence. Here, we investigated the role of unliganded thyroid hormone receptor (TR) during vertebrate development using an amphibian model. Previous studies led to the hypothesis that before production of endogenous T(3), the presence of unliganded receptor is essential for premetamorphic tadpole growth. To test this hypothesis, we generated a Xenopus laevis TR beta mutant construct ineffective for gene repression owing to impaired corepressor NCoR recruitment. Overexpression by germinal transgenesis of the mutant receptor leads to lethality during early development with numerous defects in cranio-facial and eye development. These effects correlate with TR expression profiles at these early stages. Molecular analysis of transgenic mutants reveals perturbed expression of genes involved in eye development. Finally, treatment with iopanoic acid or NH-3, modulators of thyroid hormone action, leads to abnormal eye development. In conclusion, the data reveal a role of unliganded TR in eye development.


Assuntos
Olho/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Mutação , Receptores dos Hormônios Tireóideos/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Meios de Contraste/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Ácido Iopanoico/farmacologia , Ligantes , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Receptores dos Hormônios Tireóideos/genética , Proteínas de Xenopus/genética , Xenopus laevis
17.
J Mol Evol ; 57(5): 501-14, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14738308

RESUMO

Type I collagen in tetrapods is usually a heterotrimeric molecule composed of two alpha1 and one alpha2 chains. In some teleosts, a third alpha chain has been identified by chromatography, suggesting that type I collagen should also exist as an alpha1(I)alpha2(I)alpha3(I) heterotrimer. We prepared, from zebrafish, three distinct cDNAs identified to be those of the collagen alpha1(I), alpha2(I), and alpha3(I) chains. In this study on the evolution of fibrillar collagen alpha chains and their relationships, an exhaustive phylogenetic analysis, using vertebrate fibrillar collagen sequences, showed that each alpha chain constitutes a monophyletic cluster. Results obtained with the newly isolated sequences of the zebrafish showed that the alpha3(I) chain is phylogenetically close to the alpha1(I) chain and support the hypothesis that the alpha3(I) chain arose from a duplication of the alpha1(I) gene. The duplication might occur during the duplication of the actinopterygian genome, soon after the divergence of actinopterygians and sarcopterygians, a hypothesis supported by the demonstration of a syntenic evolution between a set of fibrillar collagen genes and Hox clusters in mammals. An evolutionary scenario is proposed in which phylogenetic relationships of the alpha chains of fibrillar collagens of vertebrates could be related to Hox cluster history.


Assuntos
Colágeno Tipo I/genética , Evolução Molecular , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Genes Homeobox , Dados de Sequência Molecular , Família Multigênica , Filogenia , Alinhamento de Sequência
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