Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 23
Filtrar
1.
Dev Biol ; 2024 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-38908500

RESUMO

The ability to label proteins by fusion with genetically encoded fluorescent proteins is a powerful tool for understanding dynamic biological processes. However, current approaches for expressing fluorescent protein fusions possess drawbacks, especially at the whole organism level. Expression by transgenesis risks potential overexpression artifacts while fluorescent protein insertion at endogenous loci is technically difficult and, more importantly, does not allow for tissue-specific study of broadly expressed proteins. To overcome these limitations, we have adopted the split fluorescent protein system mNeonGreen21-10/11 (split-mNG2) to achieve tissue-specific and endogenous protein labeling in zebrafish. In our approach, mNG21-10 is expressed under a tissue-specific promoter using standard transgenesis while mNG211 is inserted into protein-coding genes of interest using CRISPR/Cas-directed gene editing. Each mNG2 fragment on its own is not fluorescent, but when co-expressed the fragments self-assemble into a fluorescent complex. Here, we report successful use of split-mNG2 to achieve differential labeling of the cytoskeleton genes tubb4b and krt8 in various tissues. We also demonstrate that by anchoring the mNG21-10 component to specific cellular compartments, the split-mNG2 system can be used to manipulate protein localization. Our approach should be broadly useful for a wide range of applications.

2.
Dev Biol ; 445(2): 170-177, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30521808

RESUMO

MEF2C is a member of the highly conserved MEF2 family of transcription factors and is a key regulator of cardiovascular development. In mice, Mef2c is expressed in the developing heart and vasculature, including the endothelium. Loss of Mef2c function in germline knockout mice leads to early embryonic demise and profound developmental abnormalities in the cardiovascular system. Previous attempts to uncover the cause of embryonic lethality by specifically disrupting Mef2c function in the heart or vasculature failed to recapitulate the global Mef2c knockout phenotype and instead resulted in relatively minor defects that did not compromise viability or result in significant cardiovascular defects. However, previous studies examined the requirement of Mef2c in the myocardial and endothelial lineages using Cre lines that begin to be expressed after the expression of Mef2c has already commenced. Here, we tested the requirement of Mef2c in the myocardial and endothelial lineages using conditional knockout approaches in mice with Cre lines that deleted Mef2c prior to onset of its expression in embryonic development. We found that deletion of Mef2c in the early myocardial lineage using Nkx2-5Cre resulted in cardiac and vascular abnormalities that were indistinguishable from the defects in the global Mef2c knockout. In contrast, early deletion of Mef2c in the vascular endothelium using an Etv2::Cre line active prior to the onset of Mef2c expression resulted in viable offspring that were indistinguishable from wild type controls with no overt defects in vascular development, despite nearly complete early deletion of Mef2c in the vascular endothelium. Thus, these studies support the idea that the requirement of MEF2C for vascular development is secondary to its requirement in the heart and suggest that the observed failure in vascular remodeling in Mef2c knockout mice results from defective heart function.


Assuntos
Sistema Cardiovascular/embriologia , Animais , Fenômenos Fisiológicos Cardiovasculares/genética , Endotélio Vascular/anormalidades , Endotélio Vascular/embriologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Cardiopatias Congênitas/embriologia , Cardiopatias Congênitas/genética , Fatores de Transcrição MEF2/deficiência , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/fisiologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Mutantes , Camundongos Transgênicos , Organogênese/genética , Organogênese/fisiologia , Gravidez
3.
Proc Natl Acad Sci U S A ; 113(40): 11237-11242, 2016 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-27647901

RESUMO

Zebrafish have a remarkable capacity to regenerate their heart. Efficient replenishment of lost tissues requires the activation of different cell types including the epicardium and endocardium. A complex set of processes is subsequently needed to support cardiomyocyte repopulation. Previous studies have identified important determinants of heart regeneration; however, to date, how revascularization of the damaged area happens remains unknown. Here, we show that angiogenic sprouting into the injured area starts as early as 15 h after injury. To analyze the role of vegfaa in heart regeneration, we used vegfaa mutants rescued to adulthood by vegfaa mRNA injections at the one-cell stage. Surprisingly, vegfaa mutants develop coronaries and revascularize after injury. As a possible explanation for these observations, we find that vegfaa mutant hearts up-regulate the expression of potentially compensating genes. Therefore, to overcome the lack of a revascularization phenotype in vegfaa mutants, we generated fish expressing inducible dominant negative Vegfaa. These fish displayed minimal revascularization of the damaged area. In the absence of fast angiogenic revascularization, cardiomyocyte proliferation did not occur, and the heart failed to regenerate, retaining a fibrotic scar. Hence, our data show that a fast endothelial invasion allows efficient revascularization of the injured area, which is necessary to support replenishment of new tissue and achieve efficient heart regeneration. These findings revisit the model where neovascularization is considered to happen concomitant with the formation of new muscle. Our work also paves the way for future studies designed to understand the molecular mechanisms that regulate fast revascularization.


Assuntos
Coração/fisiopatologia , Revascularização Miocárdica , Regeneração/fisiologia , Peixe-Zebra/fisiologia , Animais , Biomarcadores/metabolismo , Proliferação de Células , Sobrevivência Celular , Vasos Coronários/patologia , Regulação da Expressão Gênica no Desenvolvimento , Resposta ao Choque Térmico , Mutação/genética , Miócitos Cardíacos/metabolismo , Neovascularização Fisiológica , Pericárdio/patologia , Ducto Torácico/patologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
4.
Development ; 140(8): 1796-806, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23533178

RESUMO

Indirect development, in which embryogenesis gives rise to a larval form, requires that some cells retain developmental potency until they contribute to the different tissues in the adult, including the germ line, in a later, post-embryonic phase. In sea urchins, the coelomic pouches are the major contributor to the adult, but how coelomic pouch cells (CPCs) are specified during embryogenesis is unknown. Here we identify the key signaling inputs into the CPC specification network and show that the forkhead factor foxY is the first transcription factor specifically expressed in CPC progenitors. Through dissection of its cis-regulatory apparatus we determine that the foxY expression pattern is the result of two signaling inputs: first, Delta/Notch signaling activates foxY in CPC progenitors; second, Nodal signaling restricts its expression to the left side, where the adult rudiment will form, through direct repression by the Nodal target pitx2. A third signal, Hedgehog, is required for coelomic pouch morphogenesis and institution of laterality, but does not directly affect foxY transcription. Knockdown of foxY results in a failure to form coelomic pouches and disrupts the expression of virtually all transcription factors known to be expressed in this cell type. Our experiments place foxY at the top of the regulatory hierarchy underlying the specification of a cell type that maintains developmental potency.


Assuntos
Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Morfogênese/fisiologia , Células-Tronco Multipotentes/citologia , Proteína Nodal/metabolismo , Receptores Notch/metabolismo , Strongylocentrotus purpuratus/embriologia , Animais , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas Hedgehog/metabolismo , Hibridização In Situ , Morfolinos/genética , Células-Tronco Multipotentes/metabolismo , Faloidina
5.
Dev Biol ; 395(2): 379-389, 2014 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-25179465

RESUMO

Endothelin-converting enzyme-1 (Ece-1), a crucial component of the Endothelin signaling pathway, is required for embryonic development and is an important regulator of vascular tone, yet the transcriptional regulation of the ECE1 gene has remained largely unknown. Here, we define the activity and regulation of an enhancer from the human ECE1 locus in vivo. The enhancer identified here becomes active in endothelial progenitor cells shortly after their initial specification and is dependent on a conserved FOX:ETS motif, a composite binding site for Forkhead transcription factors and the Ets transcription factor Etv2, for activity in vivo. The ECE1 FOX:ETS motif is bound and cooperatively activated by FoxC2 and Etv2, but unlike other described FOX:ETS-dependent enhancers, ECE1 enhancer activity becomes restricted to arterial endothelium and endocardium by embryonic day 9.5 in transgenic mouse embryos. The ECE1 endothelial enhancer also contains an evolutionarily-conserved, consensus SOX binding site, which is required for activity in transgenic mouse embryos. Importantly, the ECE1 SOX site is bound and activated by Sox17, a transcription factor involved in endothelial cell differentiation and an important regulator of arterial identity. Moreover, the ECE1 enhancer is cooperatively activated by the combinatorial action of FoxC2, Etv2, and Sox17. Although Sox17 is required for arterial identity, few direct transcriptional targets have been identified in endothelial cells. Thus, this work has important implications for our understanding of endothelial specification and arterial subspecification.


Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Endocárdio/embriologia , Endotélio Vascular/embriologia , Fatores de Transcrição Forkhead/metabolismo , Metaloendopeptidases/metabolismo , Fatores de Transcrição SOXF/metabolismo , Fatores de Transcrição/metabolismo , Animais , Ácido Aspártico Endopeptidases/genética , Clonagem Molecular , Primers do DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Endocárdio/metabolismo , Enzimas Conversoras de Endotelina , Endotélio Vascular/metabolismo , Elementos Facilitadores Genéticos/genética , Imunofluorescência , Galactosídeos , Humanos , Indóis , Metaloendopeptidases/genética , Camundongos , Camundongos Transgênicos , Mutagênese , Fatores de Transcrição SOX/metabolismo
6.
Dev Biol ; 375(1): 92-104, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23261933

RESUMO

Specification of the non-skeletogenic mesoderm (NSM) in sea urchin embryos depends on Delta signaling. Signal reception leads to expression of regulatory genes that later contribute to the aboral NSM regulatory state. In oral NSM, this is replaced by a distinct oral regulatory state in consequence of Nodal signaling. Through regulome wide analysis we identify the homeobox gene not as an immediate Nodal target. not expression in NSM causes extinction of the aboral regulatory state in the oral NSM, and expression of a new suite of regulatory genes. All NSM specific regulatory genes are henceforth expressed exclusively, in oral or aboral domains, presaging the mesodermal cell types that will emerge. We have analyzed the regulatory linkages within the aboral NSM gene regulatory network. A linchpin of this network is gataE which as we show is a direct Gcm target and part of a feedback loop locking down the aboral regulatory state.


Assuntos
Embrião não Mamífero/metabolismo , Mesoderma/embriologia , Strongylocentrotus purpuratus/embriologia , Strongylocentrotus purpuratus/genética , Animais , Técnicas de Cultura Embrionária , Fatores de Transcrição GATA/genética , Fatores de Transcrição GATA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Morfolinos/genética , Boca/embriologia , Proteína Nodal/genética , Proteína Nodal/metabolismo , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Oligonucleotídeos Antissenso/genética , Transdução de Sinais , Strongylocentrotus purpuratus/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
7.
Dev Biol ; 382(1): 268-79, 2013 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-23933172

RESUMO

The sea urchin oral ectoderm gene regulatory network (GRN) model has increased in complexity as additional genes are added to it, revealing its multiple spatial regulatory state domains. The formation of the oral ectoderm begins with an oral-aboral redox gradient, which is interpreted by the cis-regulatory system of the nodal gene to cause its expression on the oral side of the embryo. Nodal signaling drives cohorts of regulatory genes within the oral ectoderm and its derived subdomains. Activation of these genes occurs sequentially, spanning the entire blastula stage. During this process the stomodeal subdomain emerges inside of the oral ectoderm, and bilateral subdomains defining the lateral portions of the future ciliary band emerge adjacent to the central oral ectoderm. Here we examine two regulatory genes encoding repressors, sip1 and ets4, which selectively prevent transcription of oral ectoderm genes until their expression is cleared from the oral ectoderm as an indirect consequence of Nodal signaling. We show that the timing of transcriptional de-repression of sip1 and ets4 targets which occurs upon their clearance explains the dynamics of oral ectoderm gene expression. In addition two other repressors, the direct Nodal target not, and the feed forward Nodal target goosecoid, repress expression of regulatory genes in the central animal oral ectoderm thereby confining their expression to the lateral domains of the animal ectoderm. These results have permitted construction of an enhanced animal ectoderm GRN model highlighting the repressive interactions providing precise temporal and spatial control of regulatory gene expression.


Assuntos
Ectoderma/embriologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes/genética , Ouriços-do-Mar/embriologia , Ouriços-do-Mar/genética , Análise Espaço-Temporal , Animais , Padronização Corporal/genética , Ectoderma/metabolismo , Evolução Molecular , Mesoderma/embriologia , Mesoderma/metabolismo , Modelos Genéticos , Boca/embriologia , Boca/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais/genética , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica
8.
bioRxiv ; 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38464062

RESUMO

The ability to label proteins by fusion with genetically encoded fluorescent proteins is a powerful tool for understanding dynamic biological processes. However, current approaches for expressing fluorescent protein fusions possess drawbacks, especially at the whole organism level. Expression by transgenesis risks potential overexpression artifacts while fluorescent protein insertion at endogenous loci is technically difficult and, more importantly, does not allow for tissue-specific study of broadly expressed proteins. To overcome these limitations, we have adopted the split fluorescent protein system mNeonGreen21-10/11 (split-mNG2) to achieve tissue-specific and endogenous protein labeling in zebrafish. In our approach, mNG21-10 is expressed under a tissue-specific promoter using standard transgenesis while mNG211 is inserted into protein-coding genes of interest using CRISPR/Cas-directed gene editing. Each mNG2 fragment on its own is not fluorescent, but when co-expressed the fragments self-assemble into a fluorescent complex. Here, we report successful use of split-mNG2 to achieve differential labeling of the cytoskeleton genes tubb4b and krt8 in various tissues. We also demonstrate that by anchoring the mNG21-10 component to specific cellular compartments, the split-mNG2 system can be used to manipulate protein function. Our approach should be broadly useful for a wide range of applications.

9.
Dev Biol ; 364(1): 77-87, 2012 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-22306924

RESUMO

In sea urchin embryos Delta signaling specifies non-skeletogenic mesoderm (NSM). Despite the identification of some direct targets, several aspects of Delta Notch (D/N) signaling remain supported only by circumstantial evidence. To obtain a detailed and more complete image of Delta function we followed a systems biology approach and evaluated the effects of D/N perturbation on expression levels of 205 genes up to gastrulation. This gene set includes virtually all transcription factors that are expressed in a localized fashion by mid-gastrulation, and which thus provide spatial regulatory information to the embryo. Also included are signaling factors and some pigment cell differentiation genes. We show that the number of pregastrular D/N signaling targets among these regulatory genes is small and is almost exclusively restricted to non-skeletogenic mesoderm genes. However, Delta signaling also activates foxY in the small micromeres. As is the early NSM, the small micromeres are in direct contact with Delta expressing skeletogenic mesoderm. In contrast, no endoderm regulatory genes are activated by Delta signaling even during the second phase of delta expression, when this gene is transcribed in NSM cells adjacent to the endoderm. During this phase Delta provides an ongoing input which continues to activate foxY expression in small micromere progeny. Disruption of the second phase of Delta expression specifically abolishes specification of late mesodermal derivatives such as the coelomic pouches to which the small micromeres contribute.


Assuntos
Ouriços-do-Mar/embriologia , Ouriços-do-Mar/metabolismo , Transdução de Sinais , Animais , Linhagem da Célula , Gastrulação , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Fenótipo , Ouriços-do-Mar/citologia , Transcrição Gênica
10.
Dev Biol ; 369(2): 377-85, 2012 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-22771578

RESUMO

The Nodal signaling pathway is known from earlier work to be an essential mediator of oral ectoderm specification in the sea urchin embryo, and indirectly, of aboral ectoderm specification as well. Following expression of the Nodal ligand in the future oral ectoderm during cleavage, a sequence of regulatory gene activations occur within this territory which depend directly or indirectly on nodal gene expression. Here we describe additional regulatory genes that contribute to the oral ectoderm regulatory state during specification in Strongylocentrotus purpuratus, and show how their spatial expression changes dynamically during development. By means of system wide perturbation analyses we have significantly improved current knowledge of the epistatic relations among the regulatory genes of the oral ectoderm. From these studies there emerge diverse circuitries relating downstream regulatory genes directly and indirectly to Nodal signaling. A key intermediary regulator, the role of which had not previously been discerned, is the not gene. In addition to activating several genes earlier described as targets of Nodal signaling, the not gene product acts to repress other oral ectoderm genes, contributing crucially to the bilateral spatial organization of the embryonic oral ectoderm.


Assuntos
Ligantes da Sinalização Nodal/metabolismo , Strongylocentrotus purpuratus/embriologia , Strongylocentrotus purpuratus/genética , Animais , Sequência de Bases , Padronização Corporal/genética , Padronização Corporal/fisiologia , Primers do DNA/genética , Ectoderma/embriologia , Ectoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Redes Reguladoras de Genes , Modelos Biológicos , Boca/embriologia , Boca/metabolismo , Ligantes da Sinalização Nodal/genética , Transdução de Sinais , Strongylocentrotus purpuratus/metabolismo
11.
Sci Adv ; 9(48): eadh5313, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-38019918

RESUMO

Mammals have limited capacity for heart regeneration, whereas zebrafish have extraordinary regeneration abilities. During zebrafish heart regeneration, endothelial cells promote cardiomyocyte cell cycle reentry and myocardial repair, but the mechanisms responsible for promoting an injury microenvironment conducive to regeneration remain incompletely defined. Here, we identify the matrix metalloproteinase Mmp14b as an essential regulator of heart regeneration. We identify a TEAD-dependent mmp14b endothelial enhancer induced by heart injury in zebrafish and mice, and we show that the enhancer is required for regeneration, supporting a role for Hippo signaling upstream of mmp14b. Last, we show that MMP-14 function in mice is important for the accumulation of Agrin, an essential regulator of neonatal mouse heart regeneration. These findings reveal mechanisms for extracellular matrix remodeling that promote heart regeneration.


Assuntos
Células Endoteliais , Peixe-Zebra , Animais , Camundongos , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Proliferação de Células , Regeneração , Mamíferos
12.
Zebrafish ; 18(1): 20-28, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33555975

RESUMO

Inducible gene expression systems are valuable tools for studying biological processes. We previously developed an optogenetic gene expression system called TAEL that is optimized for use in zebrafish. When illuminated with blue light, TAEL transcription factors dimerize and activate gene expression downstream of the TAEL-responsive C120 promoter. By using light as the inducing agent, the TAEL/C120 system overcomes limitations of traditional inducible expression systems by enabling fine spatial and temporal regulation of gene expression. In this study, we describe ongoing efforts to improve the TAEL/C120 system. We made modifications to both the TAEL transcriptional activator and the C120 regulatory element, collectively referred to as TAEL 2.0. We demonstrate that TAEL 2.0 consistently induces higher levels of reporter gene expression and at a faster rate, but with comparable background and toxicity as the original TAEL system. With these improvements, we were able to create functional stable transgenic lines to express the TAEL 2.0 transcription factor either ubiquitously or with a tissue-specific promoter. We demonstrate that the ubiquitous line in particular can be used to induce expression at late embryonic and larval stages, addressing a major deficiency of the original TAEL system. This improved optogenetic expression system will be a broadly useful resource for the zebrafish community.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/efeitos da radiação , Luz , Optogenética/métodos , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas/genética , Embrião não Mamífero , Genes Reporter/efeitos da radiação , Transdução de Sinais/genética , Transdução de Sinais/efeitos da radiação , Peixe-Zebra/embriologia , Peixe-Zebra/genética
13.
Curr Opin Biotechnol ; 18(4): 351-4, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17689240

RESUMO

Regulatory networks of transcription factors and signaling molecules lie at the heart of development. Their architecture implements logic functions whose execution propels cells from one regulatory state to the next, thus driving development forward. As an example of a subcircuit that translates transcriptional input into developmental output, we consider a particularly simple case, the regulatory processes underlying pigment cell formation in sea urchin embryos. The regulatory events in this process can be represented as elementary logic functions.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes/fisiologia , Ouriços-do-Mar/embriologia , Animais , Linhagem da Célula , Redes Reguladoras de Genes/genética , Modelos Biológicos , Ouriços-do-Mar/citologia , Ouriços-do-Mar/genética
14.
Biol Bull ; 214(3): 266-73, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18574103

RESUMO

The emphasis on the sequencing of genomes seems to make this task an end in itself. However, genome sequences and the genes that are predicted from them are really an opportunity to examine the biological function of the organism constructed by that genome. This point is illustrated here by examples in which the newly annotated gene complement reveals surprises about the way Strongylocentrotus purpuratus, the purple sea urchin, goes about its business. The three topics considered here are the nature of the innate immune system; the unexpected complexity of sensory function implied by genes encoding sensory proteins; and the remarkable intricacy of the regulatory gene complement in embryogenesis.


Assuntos
Genoma , Strongylocentrotus purpuratus/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento , Sistema Imunitário , Células Receptoras Sensoriais
15.
Methods Mol Biol ; 1565: 87-104, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28364236

RESUMO

The control processes that underlie the progression of development can be summarized in maps of gene regulatory networks (GRNs). A critical step in their assembly is the systematic perturbation of network candidates. In sea urchins the most important method for interfering with expression in a gene-specific way is application of morpholino antisense oligonucleotides (MOs). MOs act by binding to their sequence complement in transcripts resulting in a block in translation or a change in splicing and thus result in a loss of function. Despite the tremendous success of this technology, recent comparisons to mutants generated by genome editing have led to renewed criticism and challenged its reliability. As with all methods based on sequence recognition, MOs are prone to off-target binding that may result in phenotypes that are erroneously ascribed to the loss of the intended target. However, the slow progression of development in sea urchins has enabled extremely detailed studies of gene activity in the embryo. This wealth of knowledge paired with the simplicity of the sea urchin embryo enables careful analysis of MO phenotypes through a variety of methods that do not rely on terminal phenotypes. This article summarizes the use of MOs in probing GRNs and the steps that should be taken to assure their specificity.


Assuntos
Regulação da Expressão Gênica , Redes Reguladoras de Genes , Morfolinos/genética , Ouriços-do-Mar/genética , Animais , Pareamento de Bases , Técnicas de Silenciamento de Genes , Marcação de Genes , Técnicas de Transferência de Genes , Morfolinos/administração & dosagem , Mutação , Oligonucleotídeos Antissenso/genética , Fenótipo
16.
FEMS Microbiol Lett ; 243(1): 29-35, 2005 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-15667997

RESUMO

We have estimated the statistical distribution of the number of plasmids taken up by individual Jurkat lymphoma cells during electroporation in the presence of two plasmids, one encoding for yellow (EYFP) the other for cyan (ECFP) fluorescent protein. The plasmid concentration at which most of the cells take up only one plasmid or several molecules was determined by statistical analysis. We found that cells behaved slightly heterogeneous in plasmid uptake and describe how the homogeneity of a cell population can be quantified by Poisson statistics in order to identify experimental conditions that yield homogeneously transfection-competent cell populations. The experimental procedure worked out with Jurkat cells was applied to assay the effectiveness of antisense RNA in knocking down gene expression in Physarum polycephalum. Double transfection of flagellates with vectors encoding EYFP and antisense-EYFP revealed for the first time that gene expression can be suppressed by co-expression of antisense RNA in Physarum. Quantitative analysis revealed that one copy of antisense expressing gene per EYFP gene was sufficient to completely suppress formation of the EYFP protein in Physarum.


Assuntos
Regulação da Expressão Gênica , Células Jurkat/química , Physarum polycephalum/metabolismo , Plasmídeos/genética , RNA Antissenso/metabolismo , Transfecção , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Eletroporação , Células Eucarióticas/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Physarum polycephalum/genética , Distribuição de Poisson , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transformação Genética
17.
Gene Expr Patterns ; 10(4-5): 177-84, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20398801

RESUMO

Accurate measurements of transcript abundance are a prerequisite to understand gene activity in development. Using the NanoString nCounter, an RNA counting device, we measured the prevalence of 172 transcription factors and signaling molecules in early sea urchin development. These measurements show high fidelity over more than five orders of magnitude down to a few transcripts per embryo. Most of the genes included are locally restricted in their spatial expression, and contribute to the divergent regulatory states of cells in the developing embryo. In order to obtain high-resolution expression profiles from fertilization to late gastrulation samples were collected at hourly intervals. The measured time courses agree well with, and substantially extend, prior relative abundance measurements obtained by quantitative PCR. High temporal resolution permits sequences of successively activated genes to be precisely delineated providing an ancillary tool for assembling maps of gene regulatory networks. The data are available via an interactive website for quick plotting of selected time courses.


Assuntos
Genes Reguladores , Ouriços-do-Mar/genética , Animais , Perfilação da Expressão Gênica , Ouriços-do-Mar/crescimento & desenvolvimento
18.
Nat Protoc ; 3(12): 1876-87, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-19008874

RESUMO

Regulatory genes form large networks that are fundamental to the developmental program. The protocol presented here describes a general approach to assemble maps of gene regulatory networks (GRNs). It combines high-resolution spatio-temporal profiling of regulatory genes, strategies to perturb gene expression and quantification of perturbation effects on other genes of the network. The map of the GRN emerges by integration of these data sources and explains developmental events in terms of functional linkages between regulatory genes. This protocol has been successfully applied to regulatory processes in the sea urchin embryo, but it is generally applicable to any developmental process that relies primarily on transcriptional regulation. Unraveling the GRN for a whole tissue or organ is a challenging undertaking and, depending on the complexity, may take anywhere from months to years to complete.


Assuntos
Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Ouriços-do-Mar/genética , Animais , Embrião não Mamífero , Reação em Cadeia da Polimerase , Ouriços-do-Mar/embriologia
19.
Dev Biol ; 300(1): 485-95, 2006 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-17056028

RESUMO

The predicted gene models derived from the sea urchin genome were compared to the gene catalogs derived from other completed genomes. The models were categorized by their best match to conserved protein domains. Identification of potential orthologs and assignment of sea urchin gene models to groups of homologous genes was accomplished by BLAST alignment and through the use of a clustering algorithm. For the first time, an overview of the sea urchin genetic toolkit emerges and by extension a more precise view of the features shared among the gene catalogs that characterize the super-clades of animals: metazoans, bilaterians, chordate and non-chordate deuterostomes, ecdysozoan and lophotrochozoan protostomes. About one third of the 40 most prevalent domains in the sea urchin gene models are not as abundant in the other genomes and thus constitute expansions that are specific at least to sea urchins if not to all echinoderms. A number of homologous groups of genes previously restricted to vertebrates have sea urchin representatives thus expanding the deuterostome complement. Obversely, the absence of representatives in the sea urchin confirms a number of chordate specific inventions. The specific complement of genes in the sea urchin genome results largely from minor expansions and contractions of existing families already found in the common metazoan "toolkit" of genes. However, several striking expansions shed light on how the sea urchin lives and develops.


Assuntos
Genoma , Ouriços-do-Mar/genética , Animais , Cnidários/classificação , Cnidários/genética , Bases de Dados Genéticas , Filogenia , Proteínas/genética , Ouriços-do-Mar/classificação , Alinhamento de Sequência
20.
Dev Biol ; 300(1): 108-20, 2006 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-16997293

RESUMO

The C2H2 zinc finger is one of the most abundant protein domains and is thought to have been extensively replicated in diverse animal clades. Some well-studied proteins that contain this domain are transcriptional regulators. As part of an attempt to delineate all transcription factors encoded in the Strongylocentrotus purpuratus genome, we identified the C2H2 zinc finger genes indicated in the sequence, and examined their involvement in embryonic development. We found 377 zinc finger genes in the sea urchin genome, about half the number found in mice or humans. Their expression was measured by quantitative PCR. Up to the end of gastrulation less than a third of these genes is expressed, and about 75% of the expressed genes are maternal; both parameters distinguish these from all other classes of regulatory genes as measured in other studies. Spatial expression pattern was determined by whole mount in situ hybridization for 43 genes transcribed at a sufficient level, and localized expression was observed in diverse embryonic tissues. These genes may execute important regulatory functions in development. However, the functional meaning of the majority of this large gene family remains undefined.


Assuntos
Embrião não Mamífero/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Ouriços-do-Mar/embriologia , Ouriços-do-Mar/genética , Dedos de Zinco/genética , Animais , Perfilação da Expressão Gênica , Hibridização In Situ , Filogenia , Reação em Cadeia da Polimerase , Ouriços-do-Mar/classificação , Transcrição Gênica
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA