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1.
Dev Growth Differ ; 63(1): 93-99, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33326593

RESUMO

Neural crest (NC) cells give rise to a wide variety of cell types and tissues, such as neurons and glial cells in the peripheral nervous system. Sox2, which encodes an HMG-box transcription factor, is known to mediate pluripotency of primordial germ cells and embryonic stem (ES)/induced pluripotent stem (iPS) cells, and to regulate central nervous system development. Previous studies have revealed that Sox2 is also an important regulator of NC development. This review summarizes the well-established inhibitory roles of Sox2 in NC formation and subsequent neuronal differentiation of NC-derived cells. This review also covers recent studies suggesting additional roles for Sox2 in early NC development, neurogenesis, and glial differentiation of NC-derived cells.

2.
Nat Ecol Evol ; 4(2): 261-269, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31907383

RESUMO

Unidirectional fluid flow generated by motile cilia at the left-right organizer (LRO) breaks left-right (L-R) symmetry during early embryogenesis in mouse, frog and zebrafish. The chick embryo, however, does not require motile cilia for L-R symmetry breaking. The diversity of mechanisms for L-R symmetry breaking among vertebrates and the trigger for such symmetry breaking in non-mammalian amniotes have remained unknown. Here we examined how L-R asymmetry is established in two reptiles, Madagascar ground gecko and Chinese softshell turtle. Both of these reptiles appear to lack motile cilia at the LRO. The expression of the Nodal gene at the LRO in the reptilian embryos was found to be asymmetric, in contrast to that in vertebrates such as mouse that are dependent on cilia for L-R patterning. Two paralogues of the Nodal gene derived from an ancient gene duplication are retained and expressed differentially in cilia-dependent and cilia-independent vertebrates. The expression of these two Nodal paralogues is similarly controlled in the lateral plate mesoderm but regulated differently at the LRO. Our in-depth analysis of reptilian embryos thus suggests that mammals and non-mammalian amniotes deploy distinct strategies dependent on different Nodal paralogues for rendering Nodal activity asymmetric at the LRO.


Assuntos
Padronização Corporal , Cílios , Animais , Embrião de Galinha , Madagáscar , Camundongos , Répteis , Peixe-Zebra
3.
Biol Open ; 9(2)2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-31988094

RESUMO

In the anterior foregut (AFG) of mouse embryos, the transcription factor SOX2 is expressed in the epithelia of the esophagus and proximal branches of respiratory organs comprising the trachea and bronchi, whereas NKX2.1 is expressed only in the epithelia of respiratory organs. Previous studies using hypomorphic Sox2 alleles have indicated that reduced SOX2 expression causes the esophageal epithelium to display some respiratory organ characteristics. In the present study, we produced mouse embryos with AFG-specific SOX2 deficiency. In the absence of SOX2 expression, a single NKX2.1-expressing epithelial tube connected the pharynx and the stomach, and a pair of bronchi developed in the middle of the tube. Expression patterns of NKX2.1 and SOX9 revealed that the anterior and posterior halves of SOX2-deficient AFG epithelial tubes assumed the characteristics of the trachea and bronchus, respectively. In addition, we found that mesenchymal tissues surrounding the SOX2-deficient NKX2.1-expressing epithelial tube changed to those surrounding the trachea and bronchi in the anterior and posterior halves, as indicated by the arrangement of smooth muscle cells and SOX9-expressing cells and by the expression of Wnt4 (esophagus specific), Tbx4 (respiratory organ specific), and Hoxb6 (distal bronchus specific). The impact of mesenchyme-derived signaling on the early stage of AFG epithelial specification has been indicated. Our study demonstrated an opposite trend where epithelial tissue specification causes concordant changes in mesenchymal tissues, indicating a reciprocity of epithelial-mesenchymal interactions.

4.
Genes Cells ; 25(4): 242-256, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31997540

RESUMO

The transcription factor (TF) SOX2 regulates various stem cells and tissue progenitors via functional interactions with cell type-specific partner TFs that co-bind to enhancer sequences. Neural progenitors are the major embryonic tissues where SOX2 assumes central regulatory roles. In order to characterize the partner TFs of SOX2 in neural progenitors, we investigated the regulation of the D1 enhancer of the Sox2 gene, which is activated in the embryonic neural tube (NT) and neural crest (NC), using chicken embryo electroporation. We identified essential TF binding sites for a SOX, and two ZIC TFs in the activation of the D1 enhancer. By comparison of dorso-ventral and antero-posterior patterns of D1 enhancer activation, and the effect of mutations on the enhancer activation patterns with TF expression patterns, we determined SOX2 and ZIC2 as the major D1 enhancer-activating TFs. Binding of these TFs to the D1 enhancer sequence was confirmed by chromatin immunoprecipitation analysis. The combination of SOX2 and ZIC2 TFs activated the enhancer in both the NT and NC. These results indicate that SOX2 and ZIC2, which have been known to play major regulatory roles in neural progenitors, do functionally cooperate. In addition, the recently demonstrated SOX2 expression during the NC development is accounted for at least partly by the D1 enhancer activity. Deletion of the D1 enhancer sequence from the mouse genome, however, did not affect the mouse development, indicating functional redundancies of other enhancers.


Assuntos
Elementos Facilitadores Genéticos/genética , Crista Neural/metabolismo , Tubo Neural/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/metabolismo , Animais , Embrião de Galinha , Galinhas , Embrião de Mamíferos/metabolismo , Camundongos , Fatores de Transcrição SOXB1/genética
5.
Dev Growth Differ ; 60(3): 133-145, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29520762

RESUMO

To elucidate the transcriptional regulation that underlies specification of the otic placode, we investigated the Sox3 downstream enhancer Otic1 of the chicken, the activity of which is restricted to and distributed across the entire otic placode. The 181-bp Otic1 enhancer sequence was dissected into a 68-bp minimal activating sequence, which exhibited dimer enhancer activity in the otic placode and cephalic neural crest, and this was further reduced to a 25-bp Otic1 core sequence, which also showed octamer enhancer activity in the same regions. The Otic1 core octamer was activated by the combined action of Sall4 and the SoxE transcription factors (TFs) Sox8 or Sox9. Binding of Sall4, Sox8 and Sox9 to the Otic1 sequence in embryonic tissues was confirmed by ChIP-qPCR analysis. The core-adjoining 3' side sequences of Otic1 augmented its enhancer activity, while inclusion of the CAGGTG sequence in the immediate 3' end of the 68-bp sequence repressed its enhancer activity outside the otic placode. The CAGGTG sequence likely serves as the binding sites of the repressor TFs δEF1 (Zeb1), Sip1 (Zeb2), and Snail2, all of which are expressed in the cephalic neural crest but not in the otic placode. Therefore, the combination of Sall4-Sox8-dependent activation and CAGGTG sequence-dependent repression determines otic placode development. Although the Otic1 sequence is not conserved in mammals or fishes, the activation mechanism is, as Otic1 was also activated in otic placode tissues developed from mouse embryonic stem cells and transient transgenic zebrafish embryos.


Assuntos
Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXE/metabolismo , Animais , Galinhas , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição da Família Snail/metabolismo
6.
Dev Biol ; 433(1): 61-74, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29137924

RESUMO

Transcription factor gene Sox2 is expressed throughout sensory development, but the enhancers that regulate the gene vary depending on the developmental stages and tissues. To gain new insights into the gene regulatory network in sensory placode specification, regulation of the nasal-otic bispecific NOP1 enhancer of Sox2 was investigated in chicken embryos. Deletion and mutational analyses using electroporation showed that transcriptional repression mechanisms in combination with activation mechanisms determine placodal specificity. Activation of the NOP1 enhancer involves synergistic action by Sall4 and SoxB1/SoxE factors that bind to the adjacent sites. Deletion of repressive elements resulted in widening of the tissue area for enhancer activity to a region where the expression of Sall4 and SoxB1/E overlaps, e.g., the CNS and neural crest. Among multiple repressive elements that contribute to the placodal confinement of the NOP1 enhancer activity, CACCT/CACCTG motifs bound by Zeb/Snail family repressors play important roles. Overexpression of δEF1 (Zeb1) or Snail2 (Slug) strongly inhibited NOP1 activity. These data indicate that both activation by Sall4-Sox synergism and multiple repression mechanisms involving Zeb/Snail factors are essential for Sox2 regulation to be confined to the nasal and otic placodes.


Assuntos
Fatores de Transcrição SOXB1/metabolismo , Animais , Embrião de Galinha , Galinhas/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Crista Neural/metabolismo , Neurônios , Elementos Reguladores de Transcrição , Repressão Psicológica , Fatores de Transcrição SOXB1/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
7.
Methods Mol Biol ; 1650: 191-202, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28809022

RESUMO

Chicken embryo electroporation is a powerful tool used to identify and analyze enhancers involved in developmental gene regulation. In this chapter, the basic procedures and underlying principles of enhancer analysis using chicken embryo electroporation are described in the following steps: (1) identification of enhancers in a wide genomic region, (2) determination of the full enhancer region, (3) definition of the core enhancer regions, and (4) analysis of a functional transcription factor binding sequences in the core region.


Assuntos
Galinhas/genética , Eletroporação/métodos , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Regiões Promotoras Genéticas , Animais , Embrião de Galinha , Proteínas de Fluorescência Verde/metabolismo
8.
Dev Growth Differ ; 58(2): 205-14, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26691438

RESUMO

The vertebrate Zfhx1 transcription factor family comprises δEF1 and Sip1, which bind to CACCT-containing sequences and act as transcriptional repressors. It has been a longstanding question whether these transcription factors share the same regulatory functions in vivo. It has been shown that neural crest (NC) delamination depends on the Sip1 activity at the cranial level in mouse and chicken embryos, and it remained unclear how NC delamination is regulated at the trunk level. We observed that the expression of δEF1 and Sip1 overlaps in many tissues in chicken embryos, including NC cells at the trunk level. To clarify the above questions, we separately knocked down δEF1 and Sip1 or in combination in NC cells by electroporation of vectors expressing short hairpin RNAs (shRNAs) against respective mRNAs on the dorsal side of neural tubes that generate NC cells. In all cases, the migrating NC cell population was significantly reduced, paralleled by the decreased expression of δEF1 or Sip1 targeted by shRNAs. Expression of Sox10, the major transcription factor that regulates NC development, was also decreased by the shRNAs against δEF1 or Sip1. We conclude that the trunk NC delamination is regulated by both δEF1 and Sip1 in an analogous manner, and that these transcription factors can share equivalent regulatory functions in embryonic tissues.


Assuntos
Proteínas Aviárias/metabolismo , Movimento Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Crista Neural/embriologia , Fatores de Transcrição/metabolismo , Animais , Embrião de Galinha , Camundongos , Crista Neural/citologia
9.
Dev Growth Differ ; 57(1): 24-39, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25431100

RESUMO

The transcription factor Sox2 plays a central role in the regulation of neuro-sensory development, and many other developmental processes. To gain an in depth understanding of the Sox2 gene regulation, we previously investigated the Sox2-proximal 50-kb region of the chicken genome to determine enhancers based on functional assays using chicken embryo electroporation. We identified 11 enhancers with specificity for neuro-sensory tissues. In this study, we extended the analysis of Sox2 locus-associated enhancers to a 200-kb region and identified 16 additional enhancers with functions in neuro-sensory development. These enhancers roughly correspond to a fraction of the sequence blocks that are highly conserved between chicken and mammalian genomes. The neural enhancers were activated in sequence, thereby creating a complex pattern of functional overlaps in the developing central nervous system (CNS). The variations in the specificities of the sensory enhancers also reflected the intermediate steps of sensory tissue development. This study provides an example where a single transcription factor gene has numerous regulatory elements that allow it to fulfill many functional roles in different biological contexts.


Assuntos
Proteínas Aviárias/genética , Galinhas/genética , Mapeamento Cromossômico , Elementos Facilitadores Genéticos , Loci Gênicos/fisiologia , Genoma/fisiologia , Fatores de Transcrição SOXB1/genética , Animais
10.
Mech Dev ; 132: 59-68, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24508530

RESUMO

High expression of the B1 Sox genes, Sox2 and Sox3, is associated with the development of definitive neural primordia, the neural plates, in early stage embryos. However, in the caudal lateral epiblast (CLE) where axial stem cells reside, Sox2 and Sox3 are expressed at low levels, together with Brachyury. Because axial stem cells are the bipotential precursors of the neural plate and paraxial mesoderm, we investigated the possibility that low-level B1 Sox expression in CLE may regulate the fate of axial stem cells. We combined the genetic conditions of Sox3-null and Sox2 N1 enhancer homozygous deletion (Sox2(ΔN1/ΔN1)) to decrease B1 Sox expression in CLE. At 5-7 somite stages of mouse embryogenesis, these genetic manipulations caused approximately 30% higher production of paraxial mesodermal precursors, resulting in the development of larger somites. Analysis of mitotic cell populations suggested that decrease of B1 Sox expression in CLE does not activate cell proliferation but promotes cell migration into the mesodermal compartment. Thus, the low-level B1 Sox expression in CLE regulates axial stem cells to adjust the production of paraxial mesoderm precursors to an appropriate level.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Camadas Germinativas/metabolismo , Mesoderma/metabolismo , Placa Neural/metabolismo , Fatores de Transcrição SOXB1/genética , Animais , Desenvolvimento Embrionário/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Fatores de Transcrição SOXB1/metabolismo , Somitos/metabolismo , Células-Tronco/metabolismo
11.
J Neurosci ; 33(9): 3879-90, 2013 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-23447599

RESUMO

Sox2 is required for proper neuronal formation in the CNS, but the molecular mechanisms involved are not well characterized. Here, we addressed the role of Sox2 in neurogenesis of the developing chicken inner ear. Overexpressing Sox2 from a constitutive (ß-actin) promoter induces the expression of the proneural gene, Neurogenin1 (Ngn1); however, the expression of a downstream target of Ngn1, Neurod1, is unchanged. As a result, there is a reduction of neural precursors to delaminate and populate the developing cochleo-vestibular ganglion. In contrast, overexpression of either Ngn1 or Neurod1 is sufficient to promote the neural fate in this system. These results suggest that high levels of Sox2 inhibit progression of neurogenesis in the developing inner ear. Furthermore, we provide evidence that Ngn1 and Neurod1 inhibit Sox2 transcription through a phylogenetically conserved Sox2 enhancer to mediate neurogenesis. We propose that Sox2 confers neural competency by promoting Ngn1 expression, and that negative feedback inhibition of Sox2 by Ngn1 is an essential step in the progression from neural precursor to nascent neuron.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Orelha Interna/citologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologia , Neurônios/fisiologia , Fatores de Transcrição SOXB1/metabolismo , Fatores Etários , Animais , Animais Geneticamente Modificados , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Contagem de Células , Embrião de Galinha , Orelha Interna/embriologia , Eletroporação , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Fluorescência Verde/genética , Proteínas Luminescentes/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Inibição Neural/genética , Neurogênese/genética , Fatores de Transcrição SOXB1/genética , Tubulina (Proteína)/metabolismo
12.
PLoS One ; 7(1): e30871, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22292066

RESUMO

The proneural gene Atoh1 is crucial for the development of inner ear hair cells and it requires the function of the transcription factor Sox2 through yet unknown mechanisms. In the present work, we used the chicken embryo and HEK293T cells to explore the regulation of Atoh1 by Sox2. The results show that hair cells derive from Sox2-positive otic progenitors and that Sox2 directly activates Atoh1 through a transcriptional activator function that requires the integrity of Sox2 DNA binding domain. Atoh1 activation depends on Sox transcription factor binding sites (SoxTFBS) present in the Atoh1 3' enhancer where Sox2 directly binds, as shown by site directed mutagenesis and chromatin immunoprecipitation (ChIP). In the inner ear, Atoh1 enhancer activity is detected in the neurosensory domain and it depends on Sox2. Dominant negative competition (Sox2HMG-Engrailed) and mutation of the SoxTFBS abolish the reporter activity in vivo. Moreover, ChIP assay in isolated otic vesicles shows that Sox2 is bound to the Atoh1 enhancer in vivo. However, besides activating Atoh1, Sox2 also promotes the expression of Atoh1 negative regulators and the temporal profile of Atoh1 activation by Sox2 is transient suggesting that Sox2 triggers an incoherent feed-forward loop. These results provide a mechanism for the prosensory function of Sox2 in the inner ear. We suggest that sensory competence is established early in otic development through the activation of Atoh1 by Sox2, however, hair cell differentiation is prevented until later stages by the parallel activation of negative regulators of Atoh1 function.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Galinhas/genética , Orelha Interna/metabolismo , Orelha Interna/fisiologia , Fatores de Transcrição SOXB1/fisiologia , Sensação/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/genética , Embrião de Galinha/metabolismo , Galinhas/fisiologia , Orelha Interna/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Internas/fisiologia , Audição/genética , Audição/fisiologia , Humanos , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Sensação/fisiologia , Células-Tronco/metabolismo , Células-Tronco/fisiologia , Transfecção
13.
Biology (Basel) ; 1(3): 714-35, 2012 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-24832516

RESUMO

Development of neural and sensory primordia at the early stages of embryogenesis depends on the activity of two B1 Sox transcription factors, Sox2 and Sox3. The embryonic expression patterns of the Sox2 and Sox3 genes are similar, yet they show gene-unique features. We screened for enhancers of the 231-kb genomic region encompassing Sox3 of chicken, and identified 13 new enhancers that showed activity in different domains of the neuro-sensory primordia. Combined with the three Sox3-proximal enhancers determined previously, at least 16 enhancers were involved in Sox3 regulation. Starting from the NP1 enhancer, more enhancers with different specificities are activated in sequence, resulting in complex overlapping patterns of enhancer activities. NP1 was activated in the caudal lateral epiblast adjacent to the posterior growing end of neural plate, and by the combined action of Wnt and Fgf signaling, similar to the Sox2 N1 enhancer involved in neural/mesodermal dichotomous cell lineage segregation. The Sox3 D5 enhancer and Sox2 N3 enhancer were also activated similarly in the diencephalon, optic vesicle and lens placode, suggesting analogies in their regulation. In general, however, the specificities of the enhancers were not identical between Sox3 and Sox2, including the cases of the NP1 and D5 enhancers.

14.
Dev Growth Differ ; 53(6): 761-71, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21762129

RESUMO

Cumulative evidence now indicates pivotal roles for the group B1 Sox genes, Sox1, Sox2 and Sox3 in the genesis and development of neural primordia. Shared functions for the Sox1, Sox2 and Sox3 protein products have also been indicated. This emphasizes the importance and integral role of the group B1 Sox genes in regulating the neural primordia. We here review what is currently known about the expression patterns of both the group B1 Sox genes and the related group B2 Sox21 gene during the embryonic stages when the neural plate develops. These expression profiles are compared between the chicken and mouse embryos, both representatives of amniote species. This comparison indicates a gross conservation of the regulation of individual Sox genes, yet also demonstrates the existence of species-dependent variations, which should be taken into account when data from different species are being compared. To link the expression patterns and transcriptional regulation of these genes, contribution of gene-specific enhancers are discussed. The regulation of B1 Sox genes in the axial stem cells, the common precursors to the posterior neural plate and paraxial mesoderm and located at the posterior end of developing neural plate, is also highlighted in this review. This article thus provides a guide to performing readouts of B1/B2 Sox expression data during neural plate development in amniotes.


Assuntos
Placa Neural/citologia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB2/genética , Animais , Embrião de Galinha , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/citologia , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos , Placa Neural/embriologia , Placa Neural/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXB2/metabolismo , Especificidade da Espécie
15.
Nature ; 470(7334): 394-8, 2011 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-21331042

RESUMO

The classical view of neural plate development held that it arises from the ectoderm, after its separation from the mesodermal and endodermal lineages. However, recent cell-lineage-tracing experiments indicate that the caudal neural plate and paraxial mesoderm are generated from common bipotential axial stem cells originating from the caudal lateral epiblast. Tbx6 null mutant mouse embryos which produce ectopic neural tubes at the expense of paraxial mesoderm must provide a clue to the regulatory mechanism underlying this neural versus mesodermal fate choice. Here we demonstrate that Tbx6-dependent regulation of Sox2 determines the fate of axial stem cells. In wild-type embryos, enhancer N1 of the neural primordial gene Sox2 is activated in the caudal lateral epiblast, and the cells staying in the superficial layer sustain N1 activity and activate Sox2 expression in the neural plate. In contrast, the cells destined to become mesoderm activate Tbx6 and turn off enhancer N1 before migrating into the paraxial mesoderm compartment. In Tbx6 mutant embryos, however, enhancer N1 activity persists in the paraxial mesoderm compartment, eliciting ectopic Sox2 activation and transforming the paraxial mesoderm into neural tubes. An enhancer-N1-specific deletion mutation introduced into Tbx6 mutant embryos prevented this Sox2 activation in the mesodermal compartment and subsequent development of ectopic neural tubes, indicating that Tbx6 regulates Sox2 via enhancer N1. Tbx6-dependent repression of Wnt3a in the paraxial mesodermal compartment is implicated in this regulatory process. Paraxial mesoderm-specific misexpression of a Sox2 transgene in wild-type embryos resulted in ectopic neural tube development. Thus, Tbx6 represses Sox2 by inactivating enhancer N1 to inhibit neural development, and this is an essential step for the specification of paraxial mesoderm from the axial stem cells.


Assuntos
Linhagem da Célula , Mesoderma/citologia , Células-Tronco Neurais/citologia , Tubo Neural/citologia , Fatores de Transcrição SOXB1/metabolismo , Células-Tronco/citologia , Fatores de Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Sequência de Bases , Coristoma/embriologia , Coristoma/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Dados de Sequência Molecular , Placa Neural/citologia , Placa Neural/embriologia , Placa Neural/metabolismo , Tubo Neural/embriologia , Tubo Neural/metabolismo , Fatores de Transcrição SOXB1/genética , Proteínas com Domínio T , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Proteínas Wnt/antagonistas & inibidores , Proteínas Wnt/metabolismo , Proteína Wnt3 , Proteína Wnt3A
16.
Dev Biol ; 352(2): 354-66, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-21185279

RESUMO

The transcription factor Sox2 is a core component of the pluripotency control circuits in the early embryo, and later controls many aspects of neural development. Here, we demonstrate that Sox2 expression in the epiblast (mouse blastoderm) and anterior neural plate (ANP) is determined by the upstream enhancer N2. The mouse enhancer N2 exhibits strong activity in mouse ES cells, epiblast and ANP, and is regulated correctly in chicken and zebrafish embryos. Targeted deletion of this enhancer in mouse embryos caused a large reduction of Sox2 expression to 10% of that of wild-type levels in epiblast and ANP. However, this was tolerated by mouse embryo, probably due to functional compensation by Sox3. The activity of enhancer N2 depends on phylogenetically conserved bipartite POU factor-binding motifs in a 73-bp core sequence that function synergistically, but this activation does not involve Sox2. The major POU factor expressed at the epiblastic stage is Pou5f1 (Oct3/4), while those in the anterior neural plate are Pou3f factors (Oct6, Brn2 etc.). These factors are gradually exchanged during the transition from epiblast to ANP stages in mouse embryos and epiblast stem cells (EpiSC). Consistently, enhancer N2 activity changes from full Pou5f1 dependence to Pou3f dependence during the development of neural plate cells (NPC) from EpiSC, as assessed by specific POU factor knockdown in these cells. Zebrafish mutant embryos completely devoid of Pou5f1 activity failed to activate enhancer N2 and to express Sox2 in the blastoderm and ANP, and these defects were rescued by exogenous supply of pou5f1. Previously, Pou5f1-Sox2 synergism-dependent Sox2 activation through enhancer SRR2 in ES cells has been highlighted, but this mechanism is limited to ES cells and amniotes. In contrast, the enhancer N2-mediated, POU factor-dependent activation of Sox2, without involvement of Sox2, is a phylogenetically conserved core mechanism that functions in gene regulatory networks at early embryonic stages.


Assuntos
Camadas Germinativas/embriologia , Camadas Germinativas/metabolismo , Placa Neural/embriologia , Placa Neural/metabolismo , Fatores do Domínio POU/metabolismo , Fatores de Transcrição SOX/metabolismo , Animais , Sequência de Bases , Embrião de Galinha , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores do Domínio POU/genética , Filogenia , Fatores de Transcrição SOX/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Deleção de Sequência , Transdução de Sinais , Peixe-Zebra
17.
Dev Growth Differ ; 52(5): 397-408, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20507355

RESUMO

The development of various tissues originating from the cephalic placodes is accompanied by the expression of the Sox2 gene. This Sox2 expression initiates in the pre-placodal cephalic ectoderm, and is regulated by enhancer N-4, which also regulates Sox2 in the embryonic central nervous system (CNS) posterior to the diencephalon. As the regulation of enhancer N-4 in the ectoderm likely reflects that of the pre-placodal cell state, its regulatory elements were characterized. A 110-bp minimal and essential sequence of N-4 (mini-N-4) was determined. By mutational and deletion analyses, nine regulatory elements were determined in the mini-N-4 sequence: three elements involved in activation in both the cephalic ectoderm and CNS, three elements specifically involved in activation in the cephalic ectoderm, three elements individually involved in activation in the mesencephalon, repression in the prosencephalon, and retinoic acid response in the rhombomeric region. The cephalic ectoderm-specific elements include two potential sites for the binding of nuclear receptors, suggestive of a nuclear receptor-dependent regulation. Multimers of the 3' half of the mini-N-4 sequence, including all of the cephalic ectodermal elements, show strong and selective activity in the cephalic ectoderm, providing a powerful genetic tool for the manipulation of gene activities in the placodal lineages.


Assuntos
Sistema Nervoso Central/embriologia , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição SOXB1/genética , Animais , Sequência de Bases , Sistema Nervoso Central/metabolismo , Embrião de Galinha , Diencéfalo/embriologia , Ectoderma/metabolismo , Genes Reporter , Mesencéfalo/embriologia , Dados de Sequência Molecular , Tretinoína/farmacologia
18.
Artigo em Inglês | MEDLINE | ID: mdl-19212098

RESUMO

In higher vertebrates, the expression of Sox2, a group B1 Sox gene, is the hallmark of neural primordial cell state during the developmental processes from embryo to adult. Sox2 is regulated by the combined action of many enhancers with distinct spatio-temporal specificities. DNA sequences for these enhancers are conserved in a wide range of vertebrate species, corresponding to a majority of highly conserved non-coding sequences surrounding the Sox2 gene, corroborating the notion that the conservation of non-coding sequences mirrors their functional importance. Among the Sox2 enhancers, N-1 and N-2 are activated the earliest in embryogenesis and regulate Sox2 in posterior and anterior neural plates, respectively. These enhancers differ in their evolutionary history: the sequence and activity of enhancer N-2 is conserved in all vertebrate species, while enhancer N-1 is fully conserved only in amniotes. In teleost embryos, Sox19a/b play the major pan-neural role among the group B1 Sox paralogues, while strong Sox2 expression is limited to the anterior neural plate, reflecting the absence of posterior CNS-dedicated enhancers, including N-1. In Xenopus, neurally expressed SoxD is the orthologue of Sox19, but Sox3 appears to dominate other B1 paralogues. In amniotes, however, Sox19 has lost its group B1 Sox function and transforms into group G Sox15 (neofunctionalization), and Sox2 assumes the dominant position by gaining enhancer N-1 and other enhancers for posterior CNS. Thus, the gain and loss of specific enhancer elements during the evolutionary process reflects the change in functional assignment of particular paralogous genes, while overall regulatory functions attributed to the gene family are maintained.


Assuntos
Desenvolvimento Embrionário/genética , Evolução Molecular , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição SOXB1/genética , Homologia de Sequência do Ácido Nucleico , Animais , Sequência de Bases , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Dados de Sequência Molecular
20.
Dev Growth Differ ; 50(6): 467-74, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18422684

RESUMO

The identification of the enhancers associated with each developmentally regulated gene is a first step to clarify the regulatory mechanisms underlying embryogenesis. The electroporation technique using chicken embryo is a powerful tool to identify such enhancers. The technique enables us to survey a large genomic region and to analyze the enhancers in great detail. Comparison of the genomic sequences of the chicken and other vertebrate species identifies conserved non-coding sequence blocks to which the functionally identified enhancers often correspond. In this review, I describe in detail the methods to analyze the enhancers using the chicken embryo electroporation and genome comparison.


Assuntos
Biologia do Desenvolvimento/métodos , Eletroporação/métodos , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Animais , Embrião de Galinha , Embrião não Mamífero/metabolismo , Éxons , Técnicas Genéticas , Genoma , Proteínas de Fluorescência Verde/metabolismo , Modelos Genéticos
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