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1.
J Mol Evol ; 87(7-8): 199-208, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31332479

RESUMO

Cave animals possess remarkable phenotypes associated with existence in their dark environments. The Chinese cavefish Sinocyclocheilus tileihornes shows substantial eye degeneration, a trait shared by most cave species. The extent to which independent evolution of troglomorphic traits uses convergent molecular genetic mechanisms is as yet unknown. We performed transcriptome-wide gene expression profiling in S. tileihornes eyes and compared results with those from the closely related surface species S. angustiporus and an independently derived congeneric cavefish, S. anophthalmus. In total, 52.85 million 100 bp long paired-end clean reads were generated for S. tileihornes, and we identified differentially expressed genes between the three possible pairs of species. Functional analysis of genes differentially expressed between S. tileihornes and S. angustiporus revealed that phototransduction (KEGG id: dre04744) was the most significantly enriched pathway, indicating the obvious differences in response to captured photons between the cavefish S. tileihornes and the surface species S. angustiporus. Analysis of key genes regulating eye development showed complete absence of otx5b (orthodenticle homolog 5) expression in S. tileihornes eyes, probably related to degradation of rods, but normal expression of crx (cone-rod homeobox). The enriched pathways and Otx5 are involved in phototransduction, photoreceptor formation, and regulation of photoreceptor-related gene expression. Unlike the S. tileihornes reported here, S. anophthalmus has reduced crx and otx5 expression. These results show that different species of cavefish within the same genus that independently evolved troglodyte characteristics can have different genetic mechanisms of eye degeneration.


Assuntos
Adaptação Biológica/genética , Cyprinidae/genética , Fatores de Transcrição Otx/genética , Animais , Evolução Biológica , Cavernas , Cyprinidae/metabolismo , Evolução Molecular , Olho/metabolismo , Regulação da Expressão Gênica , Fenômenos Fisiológicos Oculares , Fatores de Transcrição Otx/biossíntese , Fatores de Transcrição Otx/metabolismo , Fenótipo , Células Fotorreceptoras de Vertebrados/metabolismo , Transcriptoma
2.
Eur J Histochem ; 61(1): 2730, 2017 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-28348423

RESUMO

OTX Homeobox genes are involved in embryonic morphogenesis and in the development of olfactory epithelium in adult. Mutations occurring in the OTX genes are reported to be associated to tumorigenisis in human. No reports correlate the expression of OTX genes and neoplasms of the nasal cavity. Thus, through immunohistochemical and Real-time PCR analysis we investigated OTX1 and OTX2 expression in the more frequent types of nasal and sinonasal tumours. Variable expression of both genes were found in normal sinonasal mucosa and in tumours. Interestingly, no expression of both OTX genes were detected in sinonasal intestinal-type adenocarcinomas; only OTX1 was found in non-intestinal-type adenocarcinomas and OTX2 was selectively expressed in olfactory neuroblastomas. In conclusion, OTX1 and OTX2 genes might have a role in the pathogenesis of different types of sinonasal neoplasms.


Assuntos
Biomarcadores Tumorais/biossíntese , Estesioneuroblastoma Olfatório/metabolismo , Regulação Neoplásica da Expressão Gênica , Cavidade Nasal/metabolismo , Proteínas de Neoplasias/biossíntese , Neoplasias Nasais/metabolismo , Fatores de Transcrição Otx/biossíntese , Adulto , Estesioneuroblastoma Olfatório/patologia , Feminino , Humanos , Masculino , Cavidade Nasal/patologia , Neoplasias Nasais/patologia , Reação em Cadeia da Polimerase em Tempo Real/métodos
3.
J Vet Sci ; 18(1): 59-65, 2017 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-27297412

RESUMO

Retinal pigment epithelium (RPE) is a major component of the eye. This highly specialized cell type facilitates maintenance of the visual system. Because RPE loss induces an irreversible visual impairment, RPE generation techniques have recently been investigated as a potential therapeutic approach to RPE degeneration. A microRNA-based technique is a new strategy for producing RPE cells from adult stem cell sources. Previously, we identified that antisense microRNA-410 (anti-miR-410) induces RPE differentiation from amniotic epithelial stem cells. In this study, we investigated RPE differentiation from umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) via anti-miR-410 treatment. We identified miR-410 as a RPE-relevant microRNA in UCB-MSCs from among 21 putative human RPE-depleted microRNAs. Inhibition of miR-410 induces overexpression of immature and mature RPE-specific factors, including MITF, LRAT, RPE65, Bestrophin, and EMMPRIN. The RPE-induced cells were able to phagocytize microbeads. Results of our microRNA-based strategy demonstrated proof-of-principle for RPE differentiation in UCB-MSCs by using anti-miR-410 treatment without the use of additional factors or exogenous transduction.


Assuntos
Diferenciação Celular/genética , MicroRNAs/metabolismo , Fatores de Transcrição Otx/biossíntese , Epitélio Pigmentado da Retina/fisiologia , cis-trans-Isomerases/biossíntese , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , Fagocitose , Epitélio Pigmentado da Retina/metabolismo
4.
J Neuropathol Exp Neurol ; 76(1): 32-38, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-28025236

RESUMO

Atypical teratoid rhabdoid tumors (ATRT) are highly malignant brain tumors of early childhood that have been regarded as a homogenous entity characterized by inactivation of the SMARCB1/INI1 or SMARCA4/BRG1 genes as the only characteristic alteration. Recent studies suggest that similar to other embryonal tumors ATRT can also be divided into subgroups based on their mRNA or methylation profiles. Using microarray-based expression analysis of 12 patient ATRT specimens we demonstrated the existence of 2 subgroups of ATRT. One subgroup is characterized by high expression of OTX2, encoding a transcription factor involved in brain development. OTX2 expression was verified by immunohistochemistry and might function as a novel therapeutic target for this fatal tumor. High expression of OTX2 as well as expression of Kir7.1/KCNJ13, TRPM3 and ENPP2, which have all previously been linked to either choroid plexus epithelium or choroid plexus tumors (CPTs), suggests a close histogenetic relation of this subgroup to CPTs.


Assuntos
Biomarcadores Tumorais/biossíntese , Neoplasias do Plexo Corióideo/metabolismo , Neoplasias do Plexo Corióideo/patologia , Fatores de Transcrição Otx/biossíntese , Tumor Rabdoide/metabolismo , Tumor Rabdoide/patologia , Teratoma/metabolismo , Teratoma/patologia , Biomarcadores Tumorais/genética , Criança , Pré-Escolar , Neoplasias do Plexo Corióideo/genética , Feminino , Humanos , Recém-Nascido , Masculino , Fatores de Transcrição Otx/genética , Tumor Rabdoide/genética , Teratoma/genética
5.
Int J Oncol ; 47(5): 1703-10, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26397460

RESUMO

The homeobox transcription factor orthodenticle homeobox 2 (OTX2) plays a critical role in very early neurogenesis, but can become oncogenic when aberrantly expressed later in life. We previously discovered its novel oncogenic role in the malignant childhood brain tumor medulloblastoma and hypothesize an oncogenic role in retinoblastoma. Primary retinoblastoma tumors and cell lines were analyzed by quantitative-PCR, immunoblotting and immunohistochemistry for OTX2. The effect of modulating OTX2 expression on tumorigenesis was tested pharmacologically and by siRNA. A lentiviral shRNA-engineered vector was used for conditional knockdown studies on tumor growth in vivo. A luciferase reporter assay was used to analyze ATRA's effect on OTX2's promoter. In this study on retinoblastoma, OTX2 was frequently amplified and/or overexpressed in primary tumors and cell lines. Knockdown of OTX2 expression by siRNA or pharmacologic inhibition by all-trans retinoic acid (ATRA) repressed OTX2 expression and cell proliferation and significantly decreased tumor growth in vivo. Loss of OTX2 expression also resulted in decreased expression of C-MYC and CRX, genes previously implicated in retinoblastoma tumorigenesis. Loss of OTX2 expression increased the phosphorylation of RB, a potential mechanism of modulating cell proliferation. Aberrant expression of OTX2 may contribute to the development of retinoblastoma. OTX2 may serve as a common transcription factor that interlinks multiple tumor-driving pathways. These results also show that OTX2 can be genetically and pharmacologically targeted, providing an exciting new therapeutic option that may be less toxic and more efficacious than current treatments.


Assuntos
Proteínas de Homeodomínio/genética , Fatores de Transcrição Otx/biossíntese , Proteínas Proto-Oncogênicas c-myc/genética , Retinoblastoma/genética , Retinoblastoma/terapia , Transdução de Sinais/genética , Transativadores/genética , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/biossíntese , Humanos , Fatores de Transcrição Otx/genética , Fosforilação , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/biossíntese , Retinoblastoma/patologia , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Transativadores/biossíntese , Tretinoína/administração & dosagem
6.
Sci Rep ; 5: 12799, 2015 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-26242611

RESUMO

Knockdown mouse models, where gene dosages can be modulated, provide valuable insights into gene function. Typically, such models are generated by embryonic stem (ES) cell-based targeted insertion, or pronuclear injection, of the knockdown expression cassette. However, these methods are associated with laborious and time-consuming steps, such as the generation of large constructs with elements needed for expression of a functional RNAi-cassette, ES-cell handling, or screening for mice with the desired knockdown effect. Here, we demonstrate that reliable knockdown models can be generated by targeted insertion of artificial microRNA (amiRNA) sequences into a specific locus in the genome [such as intronic regions of endogenous eukaryotic translation elongation factor 2 (eEF-2) gene] using the Clustered Regularly Interspaced Short Palindromic Repeats/Crispr associated 9 (CRISPR/Cas9) system. We used in vitro synthesized single-stranded DNAs (about 0.5-kb long) that code for amiRNA sequences as repair templates in CRISPR/Cas9 mutagenesis. Using this approach we demonstrate that amiRNA cassettes against exogenous (eGFP) or endogenous [orthodenticle homeobox 2 (Otx2)] genes can be efficiently targeted to a predetermined locus in the genome and result in knockdown of gene expression. We also provide a strategy to establish conditional knockdown models with this method.


Assuntos
DNA de Cadeia Simples/genética , Técnicas de Silenciamento de Genes/métodos , MicroRNAs/genética , Animais , Proteínas de Bactérias/genética , Proteína 9 Associada à CRISPR , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endonucleases/genética , Feminino , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Íntrons , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microinjeções , Mutagênese Insercional , Fatores de Transcrição Otx/biossíntese , Fatores de Transcrição Otx/genética , Interferência de RNA
7.
Stem Cell Rev Rep ; 11(3): 376-86, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25351180

RESUMO

The retinal pigment epithelium (RPE) is a highly specialized cell type located between the choroid and neural retina of the eye. RPE degeneration causes irreversible visual impairment, extending to blindness. Cell therapy has recently emerged as a potential therapeutic approach for retinal degeneration. MicroRNA-based differentiation of stem cells is a new strategy for producing tissue-specific cell types. In this study, we developed a novel microRNA-based strategy for RPE induction from human amniotic epithelial stem cells (AESCs). We identified microRNAs involved in RPE development in AESCs. Of 29 putative human RPE-relevant microRNAs, microRNA-410 (miR-410) was predicted to target multiple RPE development-relevant genes. Inhibition of miR-410 induces overexpression of immature and mature RPE-specific factors, including OTX2, RPE65, Bestrophin and EMMPRIN. These RPE-like cells were morphologically altered toward a cobblestone-like shape and were able to phagocytize microbeads. We showed that miR-410 directly regulates predicted target genes OTX2 and RPE65. Our microRNA-based strategy demonstrated RPE differentiation in AESCs by treatment of an antisense microRNA-410 (anti-miR-410), without the use of additional factors or exogenous transduction. These findings suggest that miR-410 inhibition can be a useful tool for directed cell differentiation and an attractive method for cell therapy in human retinal degenerative diseases.


Assuntos
Diferenciação Celular/genética , MicroRNAs/metabolismo , Fatores de Transcrição Otx/biossíntese , cis-trans-Isomerases/biossíntese , Líquido Amniótico/citologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , MicroRNAs/genética , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo
8.
PLoS Genet ; 10(8): e1004548, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25121599

RESUMO

In chordates, neural induction is the first step of a complex developmental process through which ectodermal cells acquire a neural identity. In ascidians, FGF-mediated neural induction occurs at the 32-cell stage in two blastomere pairs, precursors respectively of anterior and posterior neural tissue. We combined molecular embryology and cis-regulatory analysis to unveil in the ascidian Ciona intestinalis the remarkably simple proximal genetic network that controls posterior neural fate acquisition downstream of FGF. We report that the combined action of two direct FGF targets, the TGFß factor Nodal, acting via Smad- and Fox-binding sites, and the transcription factor Otx suffices to trigger ascidian posterior neural tissue formation. Moreover, we found that this strategy is conserved in the distantly related ascidian Phallusia mammillata, in spite of extreme sequence divergence in the cis-regulatory sequences involved. Our results thus highlight that the modes of gene regulatory network evolution differ with the evolutionary scale considered. Within ascidians, developmental regulatory networks are remarkably robust to genome sequence divergence. Between ascidians and vertebrates, major fate determinants, such as Otx and Nodal, can be co-opted into different networks. Comparative developmental studies in ascidians with divergent genomes will thus uncover shared ascidian strategies, and contribute to a better understanding of the diversity of developmental strategies within chordates.


Assuntos
Evolução Molecular , Redes Reguladoras de Genes , Neurogênese/genética , Proteína Nodal/genética , Fatores de Transcrição Otx/genética , Animais , Sítios de Ligação , Blastômeros , Sistema Nervoso Central/crescimento & desenvolvimento , Ciona intestinalis/genética , Ciona intestinalis/crescimento & desenvolvimento , Gástrula/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Proteína Nodal/biossíntese , Fatores de Transcrição Otx/biossíntese
9.
Cell Rep ; 7(6): 1968-81, 2014 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-24931607

RESUMO

Embryonic stem cells (ESCs) are unique in that they have the capacity to differentiate into all of the cell types in the body. We know a lot about the complex transcriptional control circuits that maintain the naive pluripotent state under self-renewing conditions but comparatively less about how cells exit from this state in response to differentiation stimuli. Here, we examined the role of Otx2 in this process in mouse ESCs and demonstrate that it plays a leading role in remodeling the gene regulatory networks as cells exit from ground state pluripotency. Otx2 drives enhancer activation through affecting chromatin marks and the activity of associated genes. Mechanistically, Oct4 is required for Otx2 expression, and reciprocally, Otx2 is required for efficient Oct4 recruitment to many enhancer regions. Therefore, the Oct4-Otx2 regulatory axis actively establishes a new regulatory chromatin landscape during the early events that accompany exit from ground state pluripotency.


Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição Otx/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular/fisiologia , Camundongos , Fator 3 de Transcrição de Octâmero/biossíntese , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição Otx/biossíntese , Fatores de Transcrição Otx/genética , Ativação Transcricional
10.
Int J Dev Biol ; 57(9-10): 779-86, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24307296

RESUMO

Neural induction and anteroposterior neural patterning occur simultaneously during Xenopus gastrulation by the inhibition of BMP and Wnt signaling, respectively. However, other processes might be necessary for determining the neural-epidermal boundary. Xenopus nodal-related-3 (Xnr3) is expressed in dorsal blastula and plays a role in neural formation. In this study, we analyzed how Xnr3 affects neural patterning to identify novel mechanisms of neural-epidermal-boundary determination. In situ hybridization revealed that ventro-animal injection with Xnr3 shifted the lateral krox20 expression domain anteriorly and reduced Otx2 expression. The mature region of Xnr3 is necessary for these effects to occur, and the pro-region accelerated them. Phalloidin labeling revealed that cells around the neural-epidermal boundary lost their slender shape following Xnr3 injection. Moreover, we analyzed the cell migration of ectodermal cells and found specific Xnr3-induced effects at the neural-epidermal boundary. These findings together suggested that Xnr3 affects anterior ectoderm migration around the neural-epidermal boundary to induce a specific neural pattern abnormality. Change of the shape of surrounding ectodermal cells and the specific migratory pattern might therefore reflect the novel mechanism of neural-epidermal boundary.


Assuntos
Padronização Corporal/genética , Encéfalo/embriologia , Epiderme/embriologia , Neurulação/genética , Fator de Crescimento Transformador beta/genética , Proteínas de Xenopus/genética , Animais , Movimento Celular/genética , Proteína 2 de Resposta de Crescimento Precoce/biossíntese , Ectoderma/embriologia , Embrião não Mamífero/metabolismo , Indução Embrionária/genética , Proteínas do Olho , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Proteínas de Homeodomínio/biossíntese , Morfolinos , Placa Neural/citologia , Fatores de Transcrição Otx/biossíntese , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Xenopus/biossíntese , Proteínas de Xenopus/metabolismo , Xenopus laevis
11.
Development ; 140(1): 43-55, 2013 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-23154415

RESUMO

Mouse embryonic stem cells (ESCs) represent the naïve ground state of the preimplantation epiblast and epiblast stem cells (EpiSCs) represent the primed state of the postimplantation epiblast. Studies have revealed that the ESC state is maintained by a dynamic mechanism characterized by cell-to-cell spontaneous and reversible differences in sensitivity to self-renewal and susceptibility to differentiation. This metastable condition ensures indefinite self-renewal and, at the same time, predisposes ESCs for differentiation to EpiSCs. Despite considerable advances, the molecular mechanism controlling the ESC state and pluripotency transition from ESCs to EpiSCs have not been fully elucidated. Here we show that Otx2, a transcription factor essential for brain development, plays a crucial role in ESCs and EpiSCs. Otx2 is required to maintain the ESC metastable state by antagonizing ground state pluripotency and promoting commitment to differentiation. Furthermore, Otx2 is required for ESC transition into EpiSCs and, subsequently, to stabilize the EpiSC state by suppressing, in pluripotent cells, the mesendoderm-to-neural fate switch in cooperation with BMP4 and Fgf2. However, according to its central role in neural development and differentiation, Otx2 is crucially required for the specification of ESC-derived neural precursors fated to generate telencephalic and mesencephalic neurons. We propose that Otx2 is a novel intrinsic determinant controlling the functional integrity of ESCs and EpiSCs.


Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Fatores de Transcrição Otx/fisiologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Genes Reporter , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fatores de Transcrição Otx/biossíntese , Fatores de Transcrição Otx/deficiência , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia
12.
Cancer Res ; 72(22): 5988-6001, 2012 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-22986744

RESUMO

The brain development transcription factor OTX2 is overexpressed and/or genomically amplified in most medulloblastomas, but the mechanistic basis for its contributions in this setting are not understood. In this study, we identified OTX2 as a transcriptional repressor and a gatekeeper of myogenic and neuronal differentiation in medulloblastoma cells. OTX2 binds to the MyoD1 core enhancer through its homeobox domain, and the remarkable repressor activity exhibited by the homeobox domain renders OTX2 transcriptionally repressive. RNA interference-mediated attenuation of OTX2 expression triggered myogenic and neuronal differentiation in vitro and prolonged the survival in an orthotopic medulloblastoma mouse model. Conversely, inducing myogenic conversion of medulloblastoma cells led to the loss of OTX2 expression. In medullomyoblastoma, a medulloblastoma subtype containing muscle elements, myogenic cells share cytogenetic signatures with the primitive tumor cells and OTX2 expression was lost in the differentiated myogenic cells. Thus, OTX2 functions via its homeobox domain as a suppressor of differentiation, and the loss of OTX2 expression is linked to the myogenesis in medullomyoblastoma. Together, our findings illustrate the origin of muscle cells in medullomyoblastomas and the oncogenic mechanism of OTX2 as a repressor of diverse differentiating potential.


Assuntos
Neoplasias Encefálicas/patologia , Meduloblastoma/patologia , Células Musculares/patologia , Neurônios/patologia , Fatores de Transcrição Otx/metabolismo , Proteínas Repressoras/metabolismo , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Células COS , Diferenciação Celular/fisiologia , Chlorocebus aethiops , Feminino , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Meduloblastoma/genética , Meduloblastoma/metabolismo , Camundongos , Camundongos Nus , Células Musculares/metabolismo , Proteína MyoD/biossíntese , Proteína MyoD/genética , Proteína MyoD/metabolismo , Neurônios/metabolismo , Fatores de Transcrição Otx/biossíntese , Fatores de Transcrição Otx/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Proteínas Repressoras/genética , Transfecção
13.
Int J Dev Biol ; 55(3): 321-6, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21710438

RESUMO

The role of somites and notochords in neuroectoderm differentiation from the embryonic ectoderm and its subsequent patterning into regional compartments along rostro-caudal and dorso-ventral axes, especially in humans, remains elusive. Here, we demonstrate the co-culture effect of somites and notochords isolated from chicken embryos on the neuronal differentiation and regional identity of an adherent culture of human embryonic stem cells (hESCs). Notochord increased the efficiency and speed of neuronal induction, whereas somites had a weak neuronal inducing effect on hESCs. However, a synergistic effect was not observed when notochords and somites were used together. Moreover, in somite and notochord co-culture groups, hESCs-derived neuronal cells expressed HOXB4, OTX2, IRX3 and PAX6, indicative of dorsal hindbrain and ventral anterior identities, respectively. Our results reveal the influence of embryonic notochord and somite co-culture in providing neuronal induction as well as rostro-caudal and dorso-ventral regional identity of hESCs-derived neuronal cells. This study provides a model through which in vivo neuronal induction events may be imitated.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Notocorda/citologia , Somitos/citologia , Animais , Embrião de Galinha , Galinhas , Técnicas de Cocultura , Indução Embrionária/fisiologia , Células-Tronco Embrionárias/metabolismo , Proteínas do Olho/biossíntese , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Humanos , Placa Neural/embriologia , Notocorda/fisiologia , Fatores de Transcrição Otx/biossíntese , Fatores de Transcrição Otx/genética , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/biossíntese , Fatores de Transcrição Box Pareados/genética , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Transdução de Sinais , Somitos/fisiologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética
14.
Mol Cancer Res ; 8(10): 1344-57, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21047732

RESUMO

The transcription factor orthodenticle homeobox 2 (OTX2) has been implicated in the pathogenesis of medulloblastoma, as it is often highly expressed and sometimes amplified in these tumors. Little is known of the downstream pathways regulated by OTX2. We therefore generated MED8A and DAOY medulloblastoma cell lines with doxycycline-inducible OTX2 expression. In both cell lines, OTX2 inhibited proliferation and induced a senescence-like phenotype with senescence-associated ß-galactosidase activity. Expression profiles of time series after OTX2 induction in MED8A showed early upregulation of cell cycle genes related to the G(2)-M phase, such as AURKA, CDC25C, and CCNG2. Paradoxically, G(1)-S phase genes such as MYC, CDK4, CDK6, CCND1, and CCND2 were strongly downregulated, in line with the observed G(1) arrest. ChIP-on-chip analyses of OTX2 binding to promoter regions in MED8A and DAOY showed a strong enrichment for binding to the G(2)-M genes, suggesting a direct activation. Their mRNA expression correlated with OTX2 expression in primary tumors, underscoring the in vivo relevance of this regulation. OTX2 induction activated the P53 pathway in MED8A, but not in DAOY, which carries a mutated P53 gene. In DAOY cells, senescence-associated secretory factors, such as interleukin-6 and insulin-like growth factor binding protein 7, were strongly upregulated after OTX2 induction. We hypothesize that the imbalance in cell cycle stimulation by OTX2 leads to cellular senescence either by activating the P53 pathway or through the induction of secretory factors. Our data indicate that OTX2 directly induces a series of cell cycle genes but requires cooperating genes for an oncogenic acceleration of the cell cycle.


Assuntos
Ciclo Celular/genética , Senescência Celular/genética , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Regulação Neoplásica da Expressão Gênica , Meduloblastoma/genética , Meduloblastoma/metabolismo , Fatores de Transcrição Otx/biossíntese , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Cerebelares/patologia , Humanos , Meduloblastoma/patologia , Fatores de Transcrição Otx/genética
15.
Neuro Oncol ; 12(7): 655-63, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20511190

RESUMO

The homeobox transcription factor OTX2 plays an essential role during embryonic brain development. It is normally silenced in the adult brain, but is overexpressed by genomic amplification or other mechanisms in the majority of medulloblastomas (MBs). Retinoic acids (RAs) can suppress OTX2 expression and inhibit MB growth. In this study, 9-cis RA most potently inhibited MB cell growth. 9-cis RA functions through the downregulation of OTX2 expression, which subsequently induces neuronal differentiation of OTX2-expressing cells. Treatment with 9-cis RA reduced the growth of D425 flank xenograft tumors in mice. In an intracranial model, however, MB tumors showed resistance to 9-cis RA treatment, and we implicated fibroblast growth factor (FGF) as a potential mediator of resistance to RA therapy. These findings suggest a mechanism for RA-mediated anti-tumor effect on OTX2-positive MB cells and indicate that therapeutic targeting of OTX2 might be effective if FGF pathway-mediated resistance can be overcome.


Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Meduloblastoma/tratamento farmacológico , Fatores de Transcrição Otx/antagonistas & inibidores , Fatores de Transcrição Otx/biossíntese , Tretinoína/uso terapêutico , Animais , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo/genética , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Meduloblastoma/metabolismo , Camundongos , Camundongos Nus , Fatores de Transcrição Otx/genética , Tretinoína/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
16.
Am J Pathol ; 175(6): 2609-17, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19893048

RESUMO

The roles in brain development. Previous studies have shown the association between OTX2 and OTX1 with anaplastic and desmoplastic medulloblastomas, respectively. Here, we investigated OTX1 and OTX2 expression in Non-Hodgkin Lymphoma (NHL) and multiple myeloma. A combination of semiquantitative RT-PCR, Western blot, and immunohistochemical analyses was used to measure OTX1 and OTX2 levels in normal lymphoid tissues and in 184 tumor specimens representative of various forms of NHL and multiple myeloma. OTX1 expression was activated in 94% of diffuse large B-cell lymphomas, in all Burkitt lymphomas, and in 90% of high-grade follicular lymphomas. OTX1 was undetectable in precursor-B lymphoblastic lymphoma, chronic lymphocytic leukemia, and in most marginal zone and mantle cell lymphomas and multiple myeloma. OTX2 was undetectable in all analyzed malignancies. Analysis of OTX1 expression in normal lymphoid tissues identified a subset of resting germinal center (GC) B cells lacking PAX5 and BCL6 and expressing cytoplasmic IgG and syndecan. About 50% of OTX1(+) GC B cells co-expressed CD10 and CD20. This study identifies OTX1 as a molecular marker for high-grade GC-derived NHL and suggests an involvement of this transcription factor in B-cell lymphomagenesis. Furthermore, OTX1 expression in a subset of normal GC B cells carrying plasma cell markers suggests its possible contribution to terminal B-cell differentiation.


Assuntos
Subpopulações de Linfócitos B/metabolismo , Linfócitos B/metabolismo , Biomarcadores Tumorais/análise , Centro Germinativo/metabolismo , Linfoma não Hodgkin/metabolismo , Fatores de Transcrição Otx/biossíntese , Western Blotting , Humanos , Imuno-Histoquímica , Mieloma Múltiplo/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
17.
J Neurosci ; 28(31): 7748-64, 2008 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-18667607

RESUMO

The diversity of cell types found within the vertebrate CNS arises in part from action of complex transcriptional programs. In the retina, the programs driving diversification of various cell types have not been completely elucidated. To investigate gene regulatory networks that underlie formation and function of one retinal circuit component, the bipolar cell, transcriptional regulation of three bipolar cell-enriched genes was analyzed. Using in vivo retinal DNA transfection and reporter gene constructs, a 200 bp Grm6 enhancer sequence, a 445 bp Cabp5 promoter sequence, and a 164 bp Chx10 enhancer sequence, were defined, each driving reporter expression specifically in distinct but overlapping bipolar cell subtypes. Bioinformatic analysis of sequences revealed the presence of potential paired-type and POU homeodomain-containing transcription factor binding sites, which were shown to be critical for reporter expression through deletion studies. The paired-type homeodomain transcription factors (TFs) Crx and Otx2 and the POU homeodomain factor Brn2 are expressed in bipolar cells and interacted with the predicted binding sequences as assessed by electrophoretic mobility shift assay. Grm6, Cabp5, and Chx10 reporter activity was reduced in Otx2 loss-of-function retinas. Endogenous gene expression of bipolar cell molecular markers was also dependent on paired-type homeodomain-containing TFs, as assessed by RNA in situ hybridization and reverse transcription-PCR in mutant retinas. Cabp5 and Chx10 reporter expression was reduced in dominant-negative Brn2-transfected retinas. The paired-type and POU homeodomain-containing TFs Otx2 and Brn2 together appear to play a common role in regulating gene expression in retinal bipolar cells.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/fisiologia , Fatores do Domínio POU/fisiologia , Células Bipolares da Retina/fisiologia , Fatores de Transcrição/fisiologia , Animais , Animais Recém-Nascidos , Feminino , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fatores de Transcrição Otx/biossíntese , Fatores de Transcrição Otx/deficiência , Fatores de Transcrição Otx/genética , Fatores do Domínio POU/biossíntese , Fatores do Domínio POU/genética , Gravidez , Ratos , Ratos Sprague-Dawley , Transativadores/biossíntese , Transativadores/deficiência , Transativadores/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética
18.
Brain Res Bull ; 73(1-3): 64-74, 2007 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-17499638

RESUMO

We investigated the expression of gamma-aminobutyric acid (GABA) in the developing rat thalamus by immunohistochemistry, using light, confocal and electron microscopy. We also examined the relationship between the expression of the homeoprotein Otx2, a transcription factor implicated in brain regionalization, and the radial and non-radial migration of early generated thalamic neurons, identified by the neuronal markers calretinin (CR) and GABA. The earliest thalamic neurons generated between embryonic days (E) 13 and 15 include those of the reticular nucleus, entirely composed by GABAergic neurons. GABA immunoreactivity appeared at E14 in immature neurons and processes laterally to the neuroepithelium of the diencephalic vesicle. The embryonic and perinatal periods were characterized by the presence of abundant GABA-immunoreactive fibers, mostly tangentially oriented, and of growth cones. At E15 and E16, GABA was expressed in radially and non-radially oriented neurons in the region of the reticular thalamic migration, between the dorsal and ventral thalamic primordia, and within the dorsal thalamus. At these embryonic stages, some CR- and GABA-immunoreactive migrating-like neurons, located in the migratory stream and in the dorsal thalamus, expressed the homeoprotein Otx2. In the perinatal period, the preponderance of GABAergic neurons was restricted to the reticular nucleus and several GABAergic fibers were still detectable throughout the thalamus. The immunolabeling of fibers progressively decreased and was no longer visible by postnatal day 10, when the adult configuration of GABA immunostaining was achieved. These results reveal the spatio-temporal features of GABA expression in the developing thalamus and suggest a novel role of Otx2 in thalamic cell migration.


Assuntos
Movimento Celular/fisiologia , Neurônios/metabolismo , Fatores de Transcrição Otx/biossíntese , Tálamo/crescimento & desenvolvimento , Ácido gama-Aminobutírico/fisiologia , Animais , Calbindina 2 , Contagem de Células , Interpretação Estatística de Dados , Imuno-Histoquímica , Microscopia Confocal , Microscopia Eletrônica , Neurônios/ultraestrutura , Fatores de Transcrição Otx/genética , Ratos , Ratos Sprague-Dawley , Proteína G de Ligação ao Cálcio S100/metabolismo , Tálamo/citologia , Tálamo/fisiologia , Fixação de Tecidos
19.
Mol Endocrinol ; 21(2): 457-71, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17095579

RESUMO

To identify regions of the mouse GnRH (mGnRH) promoter that mediate tissue-specific gene expression, transgenic mice have been generated with fragments of mGnRH promoter fused to the luciferase reporter gene. In this manuscript, we examine transgenic mice, generated with -356/+28 bp and -249/+28 bp of the mGnRH gene. Both fragments of mGnRH promoter target ovarian expression of the luciferase transgene, but neuronal luciferase activity is detected only in the mice bearing the -356-bp fragment, suggesting that the DNA sequences essential for directing neuron-specific expression of the GnRH gene are located between -356 and -249 bp. Two consensus binding sites for Otx2 were identified in this promoter region and were confirmed to be functional. EMSAs demonstrated specific binding of Otx2 to the mGnRH promoter, and overexpression of Otx2 increased transcriptional activity of the mGnRH promoter in transient transfection studies. When both Otx2 binding sites were eliminated, overexpression of Otx2 had no effect. GnRH mRNA expression in immortalized GnRH-secreting cell lines was also found to correlate with Otx2 expression. In addition, transgenic mice, bearing the 356 fragment of the mGnRH gene in which the Otx2 binding sites were eliminated, have significantly lower luciferase activity in the neonatal brain compared with mice generated with intact Otx2 binding sites. Luciferase activity was, however, still present in the ovary. Our findings provide evidence that Otx2 may have a critical role in directing tissue-specific expression of the mGnRH gene to the neuron, but not the ovary.


Assuntos
Hormônio Liberador de Gonadotropina/biossíntese , Neurônios/metabolismo , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Sítios de Ligação , Encéfalo/metabolismo , Linhagem Celular , Sequência Consenso , Feminino , Regulação da Expressão Gênica , Hormônio Liberador de Gonadotropina/genética , Luciferases/biossíntese , Luciferases/genética , Camundongos , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , Neurônios/enzimologia , Fatores de Transcrição Otx/biossíntese , Fatores de Transcrição Otx/genética , Ovário/enzimologia , RNA Mensageiro/biossíntese , Transgenes
20.
Clin Genet ; 69(6): 459-70, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16712695

RESUMO

Eye development initiates as an evagination of the early neural plate, before the closure of the neural tube. Structural malformations of the eye such as anophthalmia and microphthalmia arise very early in development. It is not surprising therefore that three of the genes currently identified to play a significant role in these developmental eye anomalies are also major players in brain development and regionalization. However, as has been emerging for a high proportion of transcriptional regulators studied, these genes have evolved to play multiple roles throughout development, and perhaps even in adult tissue maintenance. This complex spatiotemporal expression pattern requires elaborate regulatory systems which we are beginning to unravel. A major component of these complex regulatory networks is a series of cis-acting elements, highly conserved through evolution, which spread large distances from the coding region of each gene. We describe how cross regulation for PAX6, SOX2 and perhaps OTX2 has now been uncovered, pointing to the mechanisms that can fine-tune the expression of such essential developmental components. These interactions also help us understand why there is significant phenotypic overlap between mutations at these three loci.


Assuntos
Proteínas de Ligação a DNA/genética , Anormalidades do Olho/embriologia , Anormalidades do Olho/genética , Proteínas do Olho/genética , Proteínas HMGB/genética , Proteínas de Homeodomínio/genética , Fatores de Transcrição Otx/genética , Fatores de Transcrição Box Pareados/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Animais , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/fisiologia , Proteínas do Olho/biossíntese , Proteínas do Olho/fisiologia , Proteínas HMGB/biossíntese , Proteínas HMGB/fisiologia , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/fisiologia , Humanos , Fatores de Transcrição Otx/biossíntese , Fatores de Transcrição Otx/fisiologia , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/biossíntese , Fatores de Transcrição Box Pareados/fisiologia , Proteínas Repressoras/biossíntese , Proteínas Repressoras/fisiologia , Fatores de Transcrição SOXB1 , Fatores de Transcrição/biossíntese , Fatores de Transcrição/fisiologia
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