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1.
bioRxiv ; 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38979273

RESUMO

Mechanical strain substantially influences tissue shape and function in various contexts, from embryonic development to disease progression. Disruptions in these processes can result in congenital abnormalities and short-circuit mechanotransduction pathways. Manipulating strain in live tissues is crucial for understanding its impact on cellular and subcellular activities. Existing tools, such as optogenetic modulation of strain, are limited to small strain over limited distance and durations. Here, we introduce a high-strain stretcher system, the TissueTractor, designed for high-resolution spatiotemporal imaging of live tissues, enabling strain application varying from 0% to over 150%. This system is needed to unravel the intricate connections between mechanical forces and developmental processes. We demonstrated the stretcher with Xenopus laevis organotypic explants, human umbilical endothelial cells, and mouse neonatal cardiomyocytes to highlight the stretcher's adaptability. These demonstrations underscore the potential of this stretcher to deepen our understanding of the mechanical cues governing tissue dynamics and morphogenesis.

2.
Sci Rep ; 11(1): 6607, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758327

RESUMO

Gastrulation is a key event in animal embryogenesis during which germ layer precursors are rearranged and the embryonic axes are established. Cell polarization is essential during gastrulation, driving asymmetric cell division, cell movements, and cell shape changes. The furry (fry) gene encodes an evolutionarily conserved protein with a wide variety of cellular functions, including cell polarization and morphogenesis in invertebrates. However, little is known about its function in vertebrate development. Here, we show that in Xenopus, Fry plays a role in morphogenetic processes during gastrulation, in addition to its previously described function in the regulation of dorsal mesoderm gene expression. Using morpholino knock-down, we demonstrate a distinct role for Fry in blastopore closure and dorsal axis elongation. Loss of Fry function drastically affects the movement and morphological polarization of cells during gastrulation and disrupts dorsal mesoderm convergent extension, responsible for head-to-tail elongation. Finally, we evaluate a functional interaction between Fry and NDR1 kinase, providing evidence of an evolutionarily conserved complex required for morphogenesis.


Assuntos
Movimento Celular , Gastrulação , Proteínas Repressoras/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Feminino , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/genética , Proteínas de Xenopus/genética , Xenopus laevis
3.
Sci Rep ; 10(1): 17326, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33060598

RESUMO

Classical cadherins are well-known adhesion molecules responsible for physically connecting neighboring cells and signaling this cell-cell contact. Recent studies have suggested novel signaling roles for "non-junctional" cadherins (NJCads); however, the function of cadherin signaling independent of cell-cell contacts remains unknown. In this study, mesendodermal cells and tissues from gastrula stage Xenopus laevis embryos demonstrate that deletion of extracellular domains of Cadherin3 (Cdh3; formerly C-cadherin in Xenopus) disrupts contact inhibition of locomotion. In both bulk Rac1 activity assays and spatio-temporal FRET image analysis, the extracellular and cytoplasmic Cdh3 domains disrupt NJCad signaling and regulate Rac1 activity in opposing directions. Stabilization of the cytoskeleton counteracted this regulation in single cell migration assays. Our study provides novel insights into adhesion-independent signaling by Cadherin3 and its role in regulating single and collective cell migration.


Assuntos
Caderinas/fisiologia , Movimento Celular/fisiologia , Proteínas de Xenopus/fisiologia , Proteínas rac1 de Ligação ao GTP/fisiologia , Animais , Caderinas/genética , Mutação , Proteínas de Xenopus/genética , Xenopus laevis/embriologia
4.
Nat Commun ; 11(1): 665, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-32005801

RESUMO

Injury, surgery, and disease often disrupt tissues and it is the process of regeneration that aids the restoration of architecture and function. Regeneration can occur through multiple strategies including stem cell expansion, transdifferentiation, or proliferation of differentiated cells. We have identified a case of regeneration in Xenopus embryonic aggregates that restores a mucociliated epithelium from mesenchymal cells. Following disruption of embryonic tissue architecture and assembly of a compact mesenchymal aggregate, regeneration first restores an epithelium, transitioning from mesenchymal cells at the surface of the aggregate. Cells establish apico-basal polarity within 5 hours and a mucociliated epithelium within 24 hours. Regeneration coincides with nuclear translocation of the putative mechanotransducer YAP1 and a sharp increase in aggregate stiffness, and regeneration can be controlled by altering stiffness. We propose that regeneration of a mucociliated epithelium occurs in response to biophysical cues sensed by newly exposed cells on the surface of a disrupted mesenchymal tissue.


Assuntos
Epiderme/química , Epiderme/fisiologia , Xenopus laevis/embriologia , Animais , Fenômenos Biomecânicos , Epiderme/embriologia , Epitélio/química , Epitélio/embriologia , Epitélio/fisiologia , Mesoderma/química , Mesoderma/embriologia , Mesoderma/fisiologia , Regeneração , Xenopus laevis/fisiologia
5.
Development ; 145(19)2018 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-30190279

RESUMO

The large-scale movements that construct complex three-dimensional tissues during development are governed by universal physical principles. Fine-grained control of both mechanical properties and force production is crucial to the successful placement of tissues and shaping of organs. Embryos of the frog Xenopus laevis provide a dramatic example of these physical processes, as dorsal tissues increase in Young's modulus by six-fold to 80 Pascal over 8 h as germ layers and the central nervous system are formed. These physical changes coincide with emergence of complex anatomical structures, rounds of cell division, and cytoskeletal remodeling. To understand the contribution of these diverse structures, we adopt the cellular solids model to relate bulk stiffness of a solid foam to the unit size of individual cells, their microstructural organization, and their material properties. Our results indicate that large-scale tissue architecture and cell size are not likely to influence the bulk mechanical properties of early embryonic or progenitor tissues but that F-actin cortical density and composition of the F-actin cortex play major roles in regulating the physical mechanics of embryonic multicellular tissues.


Assuntos
Actinas/metabolismo , Tamanho Celular , Embrião não Mamífero/citologia , Xenopus laevis/embriologia , Animais , Fenômenos Biomecânicos , Contagem de Células , Diferenciação Celular , Módulo de Elasticidade , Embrião não Mamífero/metabolismo , Fibrilinas/metabolismo , Imageamento Tridimensional , Laminina/metabolismo , Mesoderma/citologia , Mesoderma/embriologia , Modelos Biológicos , Neurulação
6.
Curr Biol ; 27(9): 1326-1335, 2017 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-28434863

RESUMO

During early cardiogenesis, bilateral fields of mesenchymal heart progenitor cells (HPCs) move from the anterior lateral plate mesoderm to the ventral midline, undergoing a mesenchymal-to-epithelial transition (MET) en route to forming a single epithelial sheet. Through tracking of tissue-level deformations in the heart-forming region (HFR) as well as movement trajectories and traction generation of individual HPCs, we find that the onset of MET correlates with a peak in mechanical stress within the HFR and changes in HPC migratory behaviors. Small-molecule inhibitors targeting actomyosin contractility reveal a temporally specific requirement of bulk tissue compliance to regulate heart development and MET. Targeting mutant constructs to modulate contractility and compliance in the underlying endoderm, we find that MET in HPCs can be accelerated in response to microenvironmental stiffening and can be inhibited by softening. To test whether MET in HPCs was responsive to purely physical mechanical cues, we mimicked a high-stress state by injecting an inert oil droplet to generate high strain in the HFR, demonstrating that exogenously applied stress was sufficient to drive MET. MET-induced defects in anatomy result in defined functional lesions in the larval heart, implicating mechanical signaling and MET in the etiology of congenital heart defects. From this integrated analysis of HPC polarity and mechanics, we propose that normal heart development requires bilateral HPCs to undergo a critical behavioral and phenotypic transition on their way to the ventral midline, and that this transition is driven in response to the changing mechanical properties of their endoderm substrate.


Assuntos
Transição Epitelial-Mesenquimal , Coração/embriologia , Coração/fisiopatologia , Células-Tronco Mesenquimais/fisiologia , Xenopus laevis/embriologia , Animais , Endoderma/citologia , Endoderma/fisiologia , Células-Tronco Mesenquimais/citologia , Organogênese , Análise Espaço-Temporal , Estresse Fisiológico , Xenopus laevis/fisiologia
7.
Dis Model Mech ; 7(1): 93-106, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24135483

RESUMO

Dysregulated phosphatidylinositol (PI) signaling has been implicated in human gastrointestinal (GI) malignancies and inflammatory states, underlining the need to study pathophysiological roles of PI in an in vivo genetic model. Here, we study the significance of PI in GI pathophysiology using the zebrafish mutant cdipt(hi559), which lacks PI synthesis, and unravel a crucial role of PI in intestinal mucosal integrity and inflammation. The cdipt(hi559) mutants exhibit abnormal villous architecture and disorganized proliferation of intestinal epithelial cells (IECs), with pathologies reminiscent of inflammatory bowel disease (IBD), including apoptosis of goblet cells, abnormal mucosecretion, bacterial overgrowth and leukocyte infiltration. The mutant IECs exhibit vacuolation, microvillus atrophy and impaired proliferation. The cdipt(hi559) gene expression profile shows enrichment of acute phase response signaling, and the endoplasmic reticulum (ER) stress factors hspa5 and xbp1 are robustly activated in the mutant GI tissue. Temporal electron micrographic analyses reveal that PI-deficient IECs undergo sequential ER-Golgi disruption, mitochondrial depletion, macroautophagy and cell death, consistent with chronic ER-stress-mediated cytopathology. Furthermore, pharmacological induction of ER stress by inhibiting protein glycosylation or PI synthase inhibition in leukocyte-specific reporter lines replicates the cdipt(hi559) inflammatory phenotype, suggesting a fundamental role of PI metabolism and ER stress in mucosal inflammation. Antibiotics and anti-inflammatory drugs resolved the inflammation, but not the autophagic necroapoptosis of IECs, suggesting that bacterial overgrowth can exacerbate ER stress pathology, whereas persistent ER stress is sufficient to trigger inflammation. Interestingly, the intestinal phenotype was partially alleviated by chemical chaperones, suggesting their therapeutic potential. Using zebrafish genetic and pharmacological models, this study demonstrates a newly identified link between intracellular PI signaling and ER-stress-mediated mucosal inflammation. The zebrafish cdipt mutants provide a powerful tool for dissecting the fundamental mechanisms of ER-stress-mediated human GI diseases and a platform to develop molecularly targeted therapies.


Assuntos
Estresse do Retículo Endoplasmático , Inflamação/metabolismo , Mucosa Intestinal/metabolismo , Fosfatidilinositóis/metabolismo , Transdução de Sinais , Alelos , Animais , Animais Geneticamente Modificados , Apoptose , Proliferação de Células , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Regulação da Expressão Gênica , Genótipo , Glicosilação , Humanos , Mucosa Intestinal/lesões , Leucócitos/citologia , Mitocôndrias/metabolismo , Mutação , Fenótipo , Peixe-Zebra
8.
PLoS One ; 8(4): e62470, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23638093

RESUMO

Sfrp5 belongs to the family of secreted frizzled related proteins (Sfrp), secreted inhibitors of Wingless-MMTV Integration Site (Wnt) signaling, which play an important role in cancer and development. We selected sfrp5 because of its compelling expression profile in the developing endoderm in zebrafish, Danio rerio. In this study, overexpression of sfrp5 in embryos results in defects in both convergent extension (CE) by inhibition of non-canonical Wnt signaling and defects in dorsoventral patterning by inhibition of Tolloid-mediated proteolysis of the BMP inhibitor Chordin. From 25 hours post fertilization (hpf) to 3 days post fertilization (dpf), both overexpression and knockdown of Sfrp5 decrease the size of the endoderm, significantly reducing liver cell number. At 3 dpf, insulin-positive endodermal cells fail to coalesce into a single pancreatic islet. We show that Sfrp5 inhibits both canonical and non-canonical Wnt signaling during embryonic and endodermal development, resulting in endodermal abnormalities.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Trato Gastrointestinal/embriologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Organogênese , Transdução de Sinais , Proteínas Wnt/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Embrião não Mamífero/fisiologia , Endoderma/metabolismo , Fertilização , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Peptídeos e Proteínas de Sinalização Intercelular/genética , Ilhotas Pancreáticas/embriologia , Ilhotas Pancreáticas/metabolismo , Fígado/embriologia , Fígado/metabolismo , Tamanho do Órgão , Fenótipo , Metaloproteases Semelhantes a Toloide/antagonistas & inibidores , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética
9.
PLoS Genet ; 7(7): e1002179, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21814518

RESUMO

The nuclear receptor DAF-12 has roles in normal development, the decision to pursue dauer development in unfavorable conditions, and the modulation of adult aging. Despite the biologic importance of DAF-12, target genes for this receptor are largely unknown. To identify DAF-12 targets, we performed chromatin immunoprecipitation followed by hybridization to whole-genome tiling arrays. We identified 1,175 genomic regions to be bound in vivo by DAF-12, and these regions are enriched in known DAF-12 binding motifs and act as DAF-12 response elements in transfected cells and in transgenic worms. The DAF-12 target genes near these binding sites include an extensive network of interconnected heterochronic and microRNA genes. We also identify the genes encoding components of the miRISC, which is required for the control of target genes by microRNA, as a target of DAF-12 regulation. During reproductive development, many of these target genes are misregulated in daf-12(0) mutants, but this only infrequently results in developmental phenotypes. In contrast, we and others have found that null daf-12 mutations enhance the phenotypes of many miRISC and heterochronic target genes. We also find that environmental fluctuations significantly strengthen the weak heterochronic phenotypes of null daf-12 alleles. During diapause, DAF-12 represses the expression of many heterochronic and miRISC target genes, and prior work has demonstrated that dauer formation can suppress the heterochronic phenotypes of many of these target genes in post-dauer development. Together these data are consistent with daf-12 acting to ensure developmental robustness by committing the animal to adult or dauer developmental programs despite variable internal or external conditions.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Redes Reguladoras de Genes/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Animais Geneticamente Modificados , Sequência de Bases , Sítios de Ligação/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Mutação/genética
10.
PLoS One ; 6(4): e18858, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-21526205

RESUMO

In the vertebrate embryo, the kidney is derived from the intermediate mesoderm. The LIM-class homeobox transcription factor lhx1 is expressed early in the intermediate mesoderm and is one of the first genes to be expressed in the nephric mesenchyme. In this study, we investigated the role of Lhx1 in specification of the kidney field by either overexpressing or depleting lhx1 in Xenopus embryos or depleting lhx1 in an explant culture system. By overexpressing a constitutively-active form of Lhx1, we established its capacity to expand the kidney field during the specification stage of kidney organogenesis. In addition, the ability of Lhx1 to expand the kidney field diminishes as kidney organogenesis transitions to the morphogenesis stage. In a complimentary set of experiments, we determined that embryos depleted of lhx1, show an almost complete loss of the kidney field. Using an explant culture system to induce kidney tissue, we confirmed that expression of genes from both proximal and distal kidney structures is affected by the absence of lhx1. Taken together our results demonstrate an essential role for Lhx1 in driving specification of the entire kidney field from the intermediate mesoderm.


Assuntos
Padronização Corporal , Proteínas de Homeodomínio/metabolismo , Rim/citologia , Células-Tronco/citologia , Proteínas de Xenopus/metabolismo , Animais , Padronização Corporal/genética , Proliferação de Células , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Proteínas de Homeodomínio/genética , Proteínas com Homeodomínio LIM , Mesoderma/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos Antissenso/farmacologia , Técnicas de Cultura de Órgãos , Células-Tronco/metabolismo , Fatores de Tempo , Fatores de Transcrição , Xenopus/embriologia , Xenopus/genética , Proteínas de Xenopus/deficiência , Proteínas de Xenopus/genética
11.
Hepatology ; 54(2): 452-62, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21488074

RESUMO

UNLABELLED: Hepatic steatosis is the initial stage of nonalcoholic fatty liver disease (NAFLD) and may predispose to more severe hepatic disease, including hepatocellular carcinoma. Endoplasmic reticulum (ER) stress has been recently implicated as a novel mechanism that may lead to NAFLD, although the genetic factors invoking ER stress are largely unknown. During a screen for liver defects from a zebrafish insertional mutant library, we isolated the mutant cdipthi559Tg/+ (hi559). CDIPT is known to play an indispensable role in phosphatidylinositol (PtdIns) synthesis. Here we show that cdipt is expressed in the developing liver, and its disruption in hi559 mutants abrogates de novo PtdIns synthesis, resulting in hepatomegaly at 5 days postfertilization. The hi559 hepatocytes display features of NAFLD, including macrovesicular steatosis, ballooning, and necroapoptosis. Gene set enrichment of microarray profiling revealed significant enrichment of endoplasmic reticulum stress response (ERSR) genes in hi559 mutants. ER stress markers, including atf6, hspa5, calr, and xbp1, are selectively up-regulated in the mutant liver. The hi559 expression profile showed significant overlap with that of mammalian hepatic ER stress and NAFLD. Ultrastructurally, the hi559 hepatocytes display marked disruption of ER architecture with hallmarks of chronic unresolved ER stress. Induction of ER stress by tunicamycin in wild-type larvae results in a fatty liver similar to hi559, suggesting that ER stress could be a fundamental mechanism contributing to hepatic steatosis. CONCLUSION: cdipt-deficient zebrafish exhibit hepatic ER stress and NAFLD pathologies, implicating a novel link between PtdIns, ER stress, and steatosis. The tractability of hi559 mutant provides a valuable tool to dissect ERSR components, their contribution to molecular pathogenesis, and evaluation of novel therapeutics of NAFLD.


Assuntos
CDP-Diacilglicerol-Inositol 3-Fosfatidiltransferase/genética , Retículo Endoplasmático/metabolismo , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Proteínas de Membrana/genética , Fosfatidilinositóis/biossíntese , Estresse Fisiológico , Proteínas de Peixe-Zebra/genética , Animais , Fígado Gorduroso/genética , Hepatócitos/metabolismo , Mutação , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
12.
Gastroenterology ; 137(4): 1321-32, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19563808

RESUMO

BACKGROUND & AIMS: The zebrafish Danio rerio is an excellent model system for mammalian gastrointestinal development. To identify differentially regulated genes important in gastrointestinal organogenesis, we profiled the transcriptome of the zebrafish developing gastrointestinal tract. METHODS: Embryos from a transgenic zebrafish line expressing green fluorescent protein (GFP) in the developing intestine, liver, and pancreas were dissociated at 4 developmental time points, their cells sorted based on GFP expression with fluorescence-activated cell sorting (FACS), and analyzed with microarrays. To improve our analysis, we annotated the Affymetrix Zebrafish GeneChip with human orthologs. RESULTS: Transcriptional profiling showed significant differences between GFP(+) and GFP(-) cells. Up-regulated genes and pathways were consistent with mammalian gastrointestinal development, such as hepatic nuclear factor gene networks and cancer. We implicate the phosphatidylinositol 3 kinase (PI3K) pathway and show that inhibition with LY294002 causes gastrointestinal defects in zebrafish. We identified novel genes, such as the microRNAs miR-217 and miR-122, the tight junction protein claudin c, the gene fam136a, and a zebrafish tetraspanin. Novel pathways include genes containing a putative transcription factor binding sequence, GGAANCGGAANY, and a nucleolar gene network. The zebrafish microarrays also identify a set of 32 genes that may mediate the effects of gain of chromosome arm 8q in human colon, liver, and pancreatic cancers. CONCLUSIONS: We successfully combine FACS and microarray profiling to follow organogenesis throughout development. These experiments identify novel genes and pathways that probably play a role in mammalian gastrointestinal development and are potential targets for therapeutic intervention in the management of gastrointestinal disease and cancer.


Assuntos
Separação Celular , Citometria de Fluxo , Trato Gastrointestinal/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Cromossomos Humanos Par 8 , Neoplasias do Sistema Digestório/genética , Trato Gastrointestinal/embriologia , Regulação Neoplásica da Expressão Gênica , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Humanos , Hibridização In Situ , Larva/genética , MicroRNAs/metabolismo , Organogênese/genética , Reprodutibilidade dos Testes , Fatores de Tempo , Peixe-Zebra/embriologia
13.
Blood ; 110(10): 3627-36, 2007 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-17698971

RESUMO

Vascular endothelial growth factor A (VEGFA) and the type III receptor tyrosine kinase receptors (RTKs) are both required for the differentiation of endothelial cells (vasculogenesis) and for the sprouting of new capillaries (angiogenesis). We have isolated a duplicated zebrafish VegfA locus, termed VegfAb, and a duplicate RTK locus with homology to KDR/FLK1 (named Kdrb). Morpholino-disrupted VegfAb embryos develop a normal circulatory system until approximately 2 to 3 days after fertilization (dpf), when defects in angiogenesis permit blood to extravasate into many tissues. Unlike the VegfAa(121) and VegfAa(165) isoforms, the VegfAb isoforms VegfAb(171) and VegfAb(210) are not normally secreted when expressed in mammalian tissue culture cells. The Kdrb locus encodes a 1361-amino acid transmembrane receptor with strong homology to mammalian KDR. Combined knockdown of both RTKs leads to defects in vascular development, suggesting that they cooperate in mediating the vascular effects of VegfA in zebrafish development. Both VegfAa and VegfAb can individually bind and promote phosphorylation of both Flk1 (Kdra) and Kdrb proteins in vitro. Taken together, our data support a model in the zebrafish, in which duplicated VegfA and multiple type III RTKs mediate vascular development.


Assuntos
Vasos Sanguíneos/embriologia , Genes Duplicados , Homologia de Sequência , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Células CHO , Células COS , Chlorocebus aethiops , Clonagem Molecular , Cricetinae , Cricetulus , Embrião não Mamífero , Isoenzimas/genética , Modelos Biológicos , Dados de Sequência Molecular , Neovascularização Fisiológica/genética , Filogenia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologia
15.
Genetics ; 164(3): 1003-14, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12871910

RESUMO

Drosophila melanogaster males dosage compensate by twofold upregulation of the expression of genes on their single X chromosome. This process requires at least five proteins and two noncoding RNAs, roX1 and roX2, which paint the male X chromosome. We used a deletion analysis to search for functional RNA domains within roX1, assaying RNA stability, targeting of the MSL proteins to the X, and rescue of male viability in a roX1(-) roX2(-) mutant background. We found that deletion of 10% segments of the RNA did not dramatically reduce function in most cases, suggesting extensive internal redundancy. The 3' 600 nt of roX1 were most sensitive to mutations, affecting proper localization and 3' processing of the RNA. Disruption of an inverted repeat predicted to form a stem-loop structure was found partially responsible for the defects observed.


Assuntos
Mecanismo Genético de Compensação de Dose , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Expressão Gênica , Fatores de Transcrição/genética , Animais , Sequência de Bases , Northern Blotting , Primers do DNA , Teste de Complementação Genética , Hibridização In Situ , Masculino , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida
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