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1.
Trends Cell Biol ; 15(9): 453-7, 2005 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16087338

RESUMEN

Borders are essential for demarcating repeated structures such as somites during vertebrate development. Two recent articles describe roles for Integrinalpha5 and its ligand Fibronectin1 in zebrafish anterior intersomitic boundary formation and link them to Notch and Eph-Ephrin pathways in epithelialization of somite boundary cells. Together with these pathways, Integrinalpha5 and Fibronectin1 orchestrate the orderly formation of somite and later myotome borders. These studies shed light on components downstream of the periodic segmentation mechanism - the 'segmentation clock' - in somitogenesis.


Asunto(s)
Integrinas/fisiología , Somitos/fisiología , Pez Cebra/embriología , Animales , Efrinas/fisiología , Epitelio/embriología , Fibronectinas/fisiología , Proteínas de Homeodominio/fisiología , Integrina alfa5/fisiología , Proteínas del Tejido Nervioso/fisiología , Receptor Notch1/fisiología , Receptores de la Familia Eph/fisiología , Proteínas de Pez Cebra/fisiología
2.
Dev Dyn ; 238(12): 3156-67, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19882725

RESUMEN

Zebrafish and Drosophila are animal models widely used in developmental biology. High-resolution microscopy and live imaging techniques have allowed the investigation of biological processes down to the cellular level in these models. Here, using fluorescence correlation spectroscopy (FCS), we show that even processes on a molecular level can be studied in these embryos. The two animal models provide different advantages and challenges. We first characterize their autofluorescence pattern and determine usable penetration depth for FCS especially in the case of zebrafish, where tissue thickness is an issue. Next, the applicability of FCS to study molecular processes is shown by the determination of blood flow velocities with high spatial resolution and the determination of diffusion coefficients of cytosolic and membrane-bound enhanced green fluorescent protein-labeled proteins in different cell types. This work provides an approach to study molecular processes in vivo and opens up the possibility to relate these molecular processes to developmental biology questions.


Asunto(s)
Drosophila melanogaster/embriología , Desarrollo Embrionario/fisiología , Pez Cebra/embriología , Animales , Animales Modificados Genéticamente , Difusión , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Embrión no Mamífero/irrigación sanguínea , Femenino , Fluorescencia , Técnica del Anticuerpo Fluorescente/instrumentación , Técnica del Anticuerpo Fluorescente/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Modelos Biológicos , Flujo Sanguíneo Regional/fisiología , Sensibilidad y Especificidad , Espectrometría de Fluorescencia/métodos , Pez Cebra/genética , Pez Cebra/metabolismo
3.
Dev Dyn ; 238(12): 3043-3055, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19882724

RESUMEN

Somites are formed progressively from the presomitic mesoderm (PSM) in a highly regulated process according to a strict periodicity driven by an oscillatory mechanism. The Notch and Wnt pathways are key components in the regulation of this somitic oscillator and data from Xenopus and zebrafish embryos indicate that the Notch-downstream target Nrarp participates in the regulation of both activities. We have analyzed Nrarp/nrarp-a expression in the PSM of chick, mouse and zebrafish embryos, and we show that it cycles in synchrony with other Notch regulated cyclic genes. In the mouse its transcription is both Wnt- and Notch-dependent, whereas in the chick and fish embryo it is simply Notch-dependent. Despite oscillating mRNA levels, Nrarp protein does not oscillate in the PSM. Finally, neither gain nor loss of Nrarp function interferes with the normal expression of Notch-related cyclic genes.


Asunto(s)
Relojes Biológicos/fisiología , Proteínas/genética , Proteínas/metabolismo , Somitos/metabolismo , Animales , Relojes Biológicos/genética , Embrión de Pollo , Embrión de Mamíferos , Embrión no Mamífero , Femenino , Regulación del Desarrollo de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular , Ratones , Modelos Biológicos , Periodicidad , Embarazo , ARN Mensajero/metabolismo , Receptores Notch/metabolismo , Receptores Notch/fisiología , Somitos/fisiología , Pez Cebra/embriología
4.
Biophys J ; 97(2): 678-86, 2009 Jul 22.
Artículo en Inglés | MEDLINE | ID: mdl-19619483

RESUMEN

The quantification of biological interactions is very important in life sciences. Here we report for the first time, to our knowledge, the determination of a biomolecular dissociation constant (K(D)) in living zebrafish embryos at physiological protein expression levels. For that purpose, we extend the application of single wavelength fluorescence cross-correlation spectroscopy into small organisms and measure the interaction of Cdc42, a small Rho-GTPase, and IQGAP1, an actin-binding scaffolding protein. Cdc42 and IQGAP1 were labeled with monomeric red fluorescent protein and enhanced green fluorescent protein, respectively. Both fluorophores were excited at a single wavelength of 514 nm, simplifying the fluorescence spectroscopy measurements and allowing quantification. For the determination of the interaction, we used two Cdc42 mutants, the constitutively active Cdc42(G12V) which is in a predominantly GTP-bound form and the dominant-negative GDP-bound Cdc42(T17N). While Cdc42(G12V) binds to IQGAP1 with an apparent K(D) of approximately 100 nM, Cdc42(T17N) has at least a one-order-of-magnitude lower affinity for the same protein. As a comparison, we measure the same protein-protein interactions in Chinese hamster ovary cell cultures but observe significant differences in protein mobility and K(D) from the zebrafish measurements, supporting the notion that bimolecular interactions depend on the biological system under investigation and are best performed under physiologically relevant conditions.


Asunto(s)
Pez Cebra/embriología , Actinas/metabolismo , Animales , Células CHO , Calibración , Movimiento Celular , Polaridad Celular , Cricetinae , Cricetulus , Regulación del Desarrollo de la Expresión Génica , Microtúbulos/metabolismo , Modelos Biológicos , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Espectrometría de Fluorescencia , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Proteínas Activadoras de ras GTPasa/química , Proteínas Activadoras de ras GTPasa/metabolismo
5.
Dev Biol ; 323(2): 216-28, 2008 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-18789916

RESUMEN

Vertebrate skeletal muscles arise from two major types of precursor cell populations which differentiate into slow and fast fibers. Six1 homeodomain transcription factor was implicated in myogenesis in mammals, but its role in the development of different types of muscle precursors remained unclear. In zebrafish, there are two close homologs of Six1: six1a (known earlier as six1) and six1b identified in this study. Here we studied the role of six1a whose expression is initiated in the fast muscle precursor region of the forming somite. In the six1a loss-of-function conditions, initiation of myog expression was compromised in fast muscle precursors whereas myod expression appeared unaffected suggestive of six1a requirement for fast muscle differentiation. Expression of myog recovered soon, but differentiation of fast muscle proceeded abnormally. Exclusion of muscle-specific transcripts, myhz1 and tpma, from the dorsal and posterior part of somites demonstrated early abnormalities in fast muscle formation. U-shaped somites, reduced birefringence, and abnormal cell morphology were observed in morphant fast muscle upon terminal differentiation. In contrast, differentiation of slow fibers appeared largely unaffected. We conclude that Six1a plays an essential role at the onset of fast muscle differentiation.


Asunto(s)
Proteínas de Homeodominio/metabolismo , Músculos/embriología , Organogénesis , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Diferenciación Celular , Proliferación Celular , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Fibras Musculares de Contracción Rápida/citología , Fibras Musculares de Contracción Rápida/metabolismo , Especificidad de Órganos , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Somitos/citología , Somitos/embriología , Células Madre/citología , Proteínas de Pez Cebra/genética
6.
Dev Growth Differ ; 51(7): 617-37, 2009 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-19712266

RESUMEN

Localized Ca(2+) signals were consistently visualized in the formed somites of intact zebrafish embryos during the early segmentation period. Unlike the regular process of somitogenesis, these signals were stochastic in nature with respect to time and location. They did, however, occur predominantly at the medial and lateral boundaries within the formed somites. Embryos were treated with modulators of [Ca(2+)](i) to explore the signal generation mechanism and possible developmental function of the stochastic transients. Blocking elements in the phosphoinositol pathway eliminated the stochastic signals but had no obvious effect, stochastic or otherwise, on the formed somites. Such treatments did, however, result in the subsequently formed somites being longer in the mediolateral dimension. Targeted uncaging of buffer (diazo-2) or Ca(2+) (NP-ethyleneglycoltetraacetic acid [EGTA]) in the presomitic mesoderm, resulted in a regular mediolateral lengthening and shortening, respectively, of subsequently formed somites. These data suggest a requirement for IP(3) receptor-mediated Ca(2+) release during convergence cell movements in the presomitic mesoderm, which appears to have a distinct function from that of the IP(3) receptor-mediated stochastic Ca(2+) signaling in the formed somites.


Asunto(s)
Señalización del Calcio , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Somitos/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Animales , Bloqueadores de los Canales de Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Embrión no Mamífero/metabolismo , Somitos/embriología , Pez Cebra/genética
7.
BMC Dev Biol ; 7: 54, 2007 May 22.
Artículo en Inglés | MEDLINE | ID: mdl-17517144

RESUMEN

BACKGROUND: During development cell migration takes place prior to differentiation of many cell types. The chemokine receptor Cxcr4 and its ligand Sdf1 are implicated in migration of several cell lineages, including appendicular muscles. RESULTS: We dissected the role of sdf1-cxcr4 during skeletal myogenesis. We demonstrated that the receptor cxcr4a is expressed in the medial-anterior part of somites, suggesting that chemokine signaling plays a role in this region of the somite. Previous reports emphasized co-operation of Sdf1a and Cxcr4b. We found that during early myogenesis Sdf1a co-operates with the second Cxcr4 of zebrafish - Cxcr4a resulting in the commitment of myoblast to form fast muscle. Disrupting this chemokine signal caused a reduction in myoD and myf5 expression and fast fiber formation. In addition, we showed that a dimerization partner of MyoD and Myf5, E12, positively regulates transcription of cxcr4a and sdf1a in contrast to that of Sonic hedgehog, which inhibited these genes through induction of expression of id2. CONCLUSION: We revealed a regulatory feedback mechanism between cxcr4a-sdf1a and genes encoding myogenic regulatory factors, which is involved in differentiation of fast myofibers. This demonstrated a role of chemokine signaling during development of skeletal muscles.


Asunto(s)
Quimiocinas CXC/genética , Regulación del Desarrollo de la Expresión Génica , Receptores CXCR4/genética , Transducción de Señal , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Movimiento Celular , Quimiocina CXCL12 , Inmunohistoquímica , Hibridación in Situ , Desarrollo de Músculos/genética , Fibras Musculares de Contracción Rápida/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Pez Cebra/crecimiento & desarrollo
8.
Mech Dev ; 116(1-2): 227-30, 2002 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-12128230

RESUMEN

Here we present the cloning of a full-length zebrafish pdgfr-alpha cDNA as well as the expression of this gene during zebrafish embryogenesis. We show that zebrafish pdgfr-alpha mRNA is present at high levels in the fertilized egg as well as in all embryonic cells up to the end of gastrulation. Spatially restricted expression of the gene started after the onset of segmentation and is mainly localized in premigratory neural crest cells, the placodes, the anterior paraxial cells of somites and the adaxial cells of the tailbud. Transient expression of this gene was also detected in the early Kupffer's vesicle, a teleost-specific structure. Expression of the zebrafish pdgfr-alpha is both conserved as well as diverged comparing to that of other vertebrate species.


Asunto(s)
Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Pez Cebra/embriología , Pez Cebra/genética , Animales , Clonación Molecular , ADN Complementario/genética , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Cresta Neural/embriología , Cresta Neural/metabolismo , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Somitos/metabolismo
9.
Biotechniques ; 35(5): 950-4, 2003 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-14628668

RESUMEN

The zebrafish is widely used for functional studies of vertebrate genes. It is accessible to manipulations during all stages of embryogenesis because the embryo develops externally and is optically transparent. However, functional studies conducted on the zebrafish have been generally limited to the earliest phase of activity of the gene of interest, which is a limitation in studies of genes that are expressed at various stages of embryonic development. It is therefore necessary to develop methods that allow for the modulation of gene activity during later stages of zebrafish development while leaving earlier functions intact. We have successfully electroporated the green fluorescent protein (GFP) reporter gene into the neural tube of the zebrafish embryo in a unidirectional or bilateral manner. This approach can be used for the functional analysis of the late role of developmental genes in the neural tube of zebrafish embryo and larvae.


Asunto(s)
Encéfalo/embriología , Encéfalo/metabolismo , ADN/administración & dosificación , ADN/farmacocinética , Electroporación/instrumentación , Electroporación/métodos , Técnicas de Transferencia de Gen , Pez Cebra/metabolismo , Animales , ADN/genética , Electrodos , Diseño de Equipo , Análisis de Falla de Equipo , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes , Distribución Tisular , Pez Cebra/embriología , Pez Cebra/genética
10.
Mol Biol Cell ; 20(19): 4183-93, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19656853

RESUMEN

Udu has been shown to play an essential role during blood cell development; however, its roles in other cellular processes remain largely unexplored. In addition, ugly duckling (udu) mutants exhibited somite and myotome boundary defects. Our fluorescence-activated cell sorting analysis also showed that the loss of udu function resulted in defective cell cycle progression and comet assay indicated the presence of increased DNA damage in udu(tu24) mutants. We further showed that the extensive p53-dependent apoptosis in udu(tu24) mutants is a consequence of activation in the Atm-Chk2 pathway. Udu seems not to be required for DNA repair, because both wild-type and udu embryos similarly respond to and recover from UV treatment. Yeast two-hybrid and coimmunoprecipitation data demonstrated that PAH-L repeats and SANT-L domain of Udu interacts with MCM3 and MCM4. Furthermore, Udu is colocalized with 5-bromo-2'-deoxyuridine and heterochromatin during DNA replication, suggesting a role in maintaining genome integrity.


Asunto(s)
Daño del ADN , Embrión no Mamífero/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides/genética , Mutación , Proteínas de Pez Cebra/genética , Animales , Apoptosis/genética , Tipificación del Cuerpo/genética , Bromodesoxiuridina/metabolismo , Células COS , Ciclo Celular/genética , Quinasa de Punto de Control 2 , Chlorocebus aethiops , Replicación del ADN/efectos de los fármacos , Embrión no Mamífero/embriología , Factores de Unión al ADN Específico de las Células Eritroides/deficiencia , Factores de Unión al ADN Específico de las Células Eritroides/metabolismo , Citometría de Flujo , Regulación del Desarrollo de la Expresión Génica , Heterocromatina/metabolismo , Inmunohistoquímica , Hibridación in Situ , Microscopía Fluorescente , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Técnicas del Sistema de Dos Híbridos , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/metabolismo
11.
Curr Biol ; 19(19): 1616-22, 2009 Oct 13.
Artículo en Inglés | MEDLINE | ID: mdl-19747827

RESUMEN

Recent studies have begun to elucidate how the endothelial lineage is specified from the nascent mesoderm. However, the molecular mechanisms which regulate this process remain largely unknown. We hypothesized that Notch signaling might play an important role in specifying endothelial progenitors from the mesoderm, given that this pathway acts as a bipotential cell-fate switch on equipotent progenitor populations in other settings. We found that zebrafish embryos with decreased levels of Notch signaling exhibited a significant increase in the number of endothelial cells, whereas embryos with increased levels of Notch signaling displayed a reduced number of endothelial cells. Interestingly, there is a concomitant gain of endothelial cells and loss of erythrocytes in embryos with decreased Notch activity, without an effect on cell proliferation or apoptosis. Lineage-tracing analyses indicate that the ectopic endothelial cells in embryos with decreased Notch activity originate from mesodermal cells that normally produce erythrocyte progenitors. Taken together, our data suggest that Notch signaling negatively regulates the number of endothelial cells by limiting the number of endothelial progenitors within the mesoderm, probably functioning as a cell-fate switch between the endothelial and the hematopoietic lineages.


Asunto(s)
Diferenciación Celular/fisiología , Linaje de la Célula/fisiología , Embrión no Mamífero/fisiología , Células Endoteliales/fisiología , Mesodermo/embriología , Receptores Notch/metabolismo , Transducción de Señal/fisiología , Pez Cebra/embriología , Análisis de Varianza , Animales , Bromodesoxiuridina , Dimetilsulfóxido , Dipéptidos , Embrión no Mamífero/citología , Inmunohistoquímica , Hibridación in Situ , Receptores Notch/fisiología , Ácido gamma-Aminobutírico/análogos & derivados
12.
Dev Dyn ; 234(4): 1055-63, 2005 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-16252281

RESUMEN

The inhibitor of differentiation or inhibitor of DNA binding (Id) family are members of the helix-loop-helix (HLH) group of transcription factors that play important roles in cell proliferation, differentiation, cell cycle control, and apoptosis. They modulate the formation of active class A-class B basic HLH (bHLH) complexes. Ids lack the amino-terminal associated basic region necessary for DNA binding, thus sequestering the class A factors, inhibiting the formation of active class A-class B heterodimers and, therefore, are considered to act as dominant-negative regulators of differentiation pathways. We isolated zebrafish id2, and its expression during development was characterized. id2, in addition to regions of expression detected in Xenopus and mice, is also expressed in the tegmentum; midbrain-hindbrain boundary; cerebellum; rhombomeres 2,3,4,6; notochord; and corpuscles of Stannius. Furthermore, we show that expression of id2 is repressed in mind bomb mutants, suggesting a role of Notch upstream of Id2.


Asunto(s)
Encéfalo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Transducción de Señal/fisiología , Pez Cebra/embriología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Análisis por Conglomerados , Crioultramicrotomía , Cartilla de ADN , ADN Complementario/genética , Proteína 2 Inhibidora de la Diferenciación/genética , Datos de Secuencia Molecular , Receptores Notch/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Transducción de Señal/genética , Especificidad de la Especie
13.
Dev Dyn ; 230(4): 781-6, 2004 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-15254912

RESUMEN

Drosophila sine oculis homologous genes in vertebrates are homeobox-containing transcription factors functioning within the Pax-Six-Eya-Dach regulatory network during development. In this study, we describe the cloning and expression of a zebrafish homolog of sine oculis, six1. The reverse transcription-polymerase chain reaction demonstrated accumulation of six1 transcripts at mid-gastrula, and in situ hybridization showed their subsequent expression in the cranial placode and later in the olfactory, otic, and lateral line placodes, inner ear, and neuromasts. In addition, six1 is expressed in the pituitary, branchial arches, somites, pectoral fin, ventral abdomen muscle, and the cranial muscles of the eye and lower jaw. An increase of six1 expression was observed in the lateral line, muscles, and inner ear of the mind bomb mutant, illustrating a regulatory effect of the Notch pathway on expression of Six genes.


Asunto(s)
Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/fisiología , Proteínas de Pez Cebra/biosíntesis , Proteínas de Pez Cebra/fisiología , Secuencia de Aminoácidos , Animales , Clonación Molecular , Drosophila , Proteínas de Drosophila , Oído/embriología , Etiquetas de Secuencia Expresada , Gástrula/metabolismo , Hibridación in Situ , Proteínas de la Membrana/fisiología , Datos de Secuencia Molecular , Desarrollo de Músculos , Músculos/embriología , Mutación , Filogenia , ARN Mensajero/metabolismo , Receptores Notch , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Factores de Tiempo , Distribución Tisular , Pez Cebra
14.
Dev Dyn ; 227(1): 14-26, 2003 May.
Artículo en Inglés | MEDLINE | ID: mdl-12701095

RESUMEN

A 1,934-bp muscle-specific promoter from the zebrafish mylz2 gene was isolated and characterized by transgenic analysis. By using a series of 5' promoter deletions linked to the green fluorescent protein (gfp) reporter gene, transient transgenic analysis indicated that the strength of promoter activity appeared to correlate to the number of muscle cis-elements in the promoter and that a minimal -77-bp region was sufficient for a relatively strong promoter activity in muscle cells. Stable transgenic lines were obtained from several mylz2-gfp constructs. GFP expression in the 1,934-bp promoter transgenic lines mimicked well the expression pattern of endogenous mylz2 mRNA in both somitic muscle and nonsomitic muscles, including fin, eye, jaw, and gill muscles. An identical pattern of GFP expression, although at a much lower level, was observed from a transgenic line with a shorter 871-bp promoter. Our observation indicates that there is no distinct cis-element for activation of mylz2 in different skeletal muscles. Furthermore, RNA encoding a dominant negative form of cAMP-dependent protein kinase A was injected into mylz2-gfp transgenic embryos and GFP expression was significantly reduced due to an expanded slow muscle development at the expense of GFP-expressing fast muscle. The mylz2-gfp transgene was also transferred into two zebrafish mutants, spadetail and chordino, and several novel phenotypes in muscle development in these mutants were discovered.


Asunto(s)
Animales Modificados Genéticamente/embriología , Proteínas Luminiscentes/metabolismo , Músculo Esquelético/crecimiento & desarrollo , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Pez Cebra/genética , Animales , Animales Modificados Genéticamente/anatomía & histología , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/crecimiento & desarrollo , Secuencia de Bases , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Fluorescentes Verdes , Hibridación in Situ , Proteínas Luminiscentes/genética , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Pez Cebra , Proteínas de Pez Cebra/metabolismo
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