RESUMEN
OBJECTIVES: As a follow-up to our previous reports showing that the G9a histone methyltransferase-specific inhibitor BIX01294 enhances bone marrow cell cardiac potential, this drug was examined for its effects on cardiomyocytes and mouse cardiac progenitor cells (CPCs). MATERIALS AND METHODS: Cardiomyocytes and cardiac explants were cultured ± BIX01294, and examined for changes in cardiac function, protein and gene expression. Additionally, enriched populations of CPCs, contained in the 'phase bright cell' component of explants, were harvested from non-treated and BIX01294-treated cardiac tissue, and assayed for differences in cell phenotype and differentiation potential. Mouse CPCs were cultured with rat cardiomyocytes to allow differentiation of the progenitors to be assayed using species-specific PCR primers. RESULTS: While BIX01294 had no discernible effect on myocyte function and sarcomeric organization, treatment with this drug significantly increased CPC proliferation, as indicated by enhanced MTT metabolization and BrdUrd incorporation (4.1- and 2.0-fold, respectively, P < 0.001) after 48 h labelling, and increased Ki67 expression (4.8-fold, P < 0.001) after 7 days culture. Heart explants exposed to BIX01294 generated 3.6-fold (P < 0.005) greater yields of CPCs by 2 weeks culture. Importantly, CPCs obtained from non-treated and BIX01294-treated cultures did not differ in phenotype or differentiation potential. CONCLUSIONS: These data indicate that BIX01294 can expand CPCs without undermining their capacity as cardiac progenitors, and suggest that this drug may have utility for generating large numbers of CPCs for cardiac repair.
Asunto(s)
Células Madre Adultas/efectos de los fármacos , Azepinas/farmacología , Inhibidores Enzimáticos/farmacología , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Miocitos Cardíacos/efectos de los fármacos , Quinazolinas/farmacología , Células Madre Adultas/citología , Células Madre Adultas/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Técnicas de Cocultivo , N-Metiltransferasa de Histona-Lisina/metabolismo , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Ratas WistarRESUMEN
WNT proteins compose a family of secreted signaling molecules that regulate cell fate and behavior. The possible influence of WNTs on hematopoietic cell fate was examined. Both hematopoietic progenitor cell (HPC)-enriched embryonic avian bone marrow cells and the quail mesodermal stem cell line QCE6 were used for these studies. Under optimized conditions, the bone marrow and QCE6 cells behaved identically and developed into red blood cells (RBCs), monocytes, macrophages, granulocytes, and thrombocytes. This broad range of blood cell phenotypes exhibited by QCE6 cells was dependent on their active expression of WNT11. However, when QCE6 cells were prevented from producing WNT11-by expression of a stably transfected WNT11 antisense transgene-the cultures were dominated by highly vacuolated macrophages. RBCs were absent from these cultures, and the presence of monocytes was greatly diminished. Exposure of these WNT11 antisense cells to soluble WNT11 or WNT5a restored the broad range of blood cell phenotypes exhibited by parental QCE6 cells. Overexpression of WNT protein in QCE6 cells further increased the prevalence of RBCs and monocytes and greatly diminished the appearance of macrophages. Accordingly, treatment of HPC-enriched bone marrow cultures with soluble WNT11 or WNT5a inhibited macrophage formation. Instead, monocytes and RBCs were the prevalent cells displayed by WNT-treated bone marrow cultures. Together, these data indicate that WNTs may play a major role in regulating hematopoietic cell fate.
Asunto(s)
Glicoproteínas/fisiología , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Proteínas Proto-Oncogénicas/fisiología , Transducción de Señal , Animales , Médula Ósea/embriología , Bovinos/sangre , Diferenciación Celular , Línea Celular , Separación Celular , Células Cultivadas/efectos de los fármacos , Pollos/sangre , Coturnix/embriología , Medios de Cultivo/farmacología , Medios de Cultivo Condicionados , ADN sin Sentido/genética , Embrión no Mamífero/citología , Eritrocitos/citología , Gástrula/citología , Regulación del Desarrollo de la Expresión Génica , Glicoproteínas/genética , Glicoproteínas/farmacología , Células Madre Hematopoyéticas/clasificación , Macrófagos/ultraestructura , Mesodermo/citología , Monocitos/citología , Fenotipo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/farmacología , Proteínas Recombinantes de Fusión/fisiología , Especificidad de la Especie , Transfección , Proteínas Wnt , Proteína Wnt-5aRESUMEN
Transition from symmetry to asymmetry is a central theme in cell and developmental biology. In Xenopus embryos, dorsal-ventral asymmetry is initiated by a microtubule-dependent cytoplasmic rotation during the first cell cycle after fertilization. Here we show that the cytoplasmic rotation initiates differential cytoplasmic polyadenylation of maternal Xwnt-11 RNA, encoding a member of the Wnt family of cell-cell signaling factors. Translational regulation of Xwnt-11 mRNA along the dorsal-ventral axis results in asymmetric accumulation of Xwnt-11 protein. These results demonstrate spatially regulated translation of a maternal cell-signaling factor along the vertebrate dorsal-ventral axis and represent a novel mechanism for Wnt gene regulation. Spatial regulation of maternal RNA translation, which has been established in invertebrates, appears to be an evolutionarily conserved mechanism in the generation of intracellular asymmetry and the consequential formation of the multicellular body pattern.
Asunto(s)
Tipificación del Cuerpo , Regulación del Desarrollo de la Expresión Génica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Procesamiento Postranscripcional del ARN , Xenopus/embriología , Animales , Ciclo Celular/genética , Citoplasma/metabolismo , Femenino , Fertilización , Modelos Genéticos , Poli A/genética , Reacción en Cadena de la Polimerasa , Polirribosomas/genética , Biosíntesis de Proteínas , ARN/análisis , Transducción de Señal , Factores de Tiempo , Factor de Crecimiento Transformador beta , Proteínas Wnt , Proteínas de XenopusRESUMEN
Cardiac tissue in the bird is derived from paired regions of lateral mesoderm within the anterior half of the embryo (Rawles [1943] Physiol. Zool. 16:22-42; Stalsberg and DeHaan [1969] Dev. Biol. 19:128-159). Previously, we reported that WNT11 is expressed in early avian mesoderm in a pattern that overlaps with the precardiac regions. To examine whether this molecule may play a role in promoting cardiogenesis, we cultured tissue explants from microdissected HH stage 4, 5, and 6 quail embryos. The isolated tissue consisted of both the mesoderm and endoderm layers from either anterior precardiac or posterior noncardiogenic regions of the embryo. As a necessary control for examining the ability of WNT11 to convert noncardiogenic mesoderm to cardiac tissue, we compared the cardiogenic potential of anterior and posterior regions. For stages 5 and 6, our results were consistent with what has been previously reported (Rawles [1943] Physiol. Zool. 16:22-42; Sugi and Lough [1994] Dev. Dyn. 200:155-162); as anterior mesoderm becomes contractile, while posterior mesoderm does not produce cardiac tissue. Surprisingly, when we examined stage 4 embryos both anterior and posterior regions gave rise to cardiac tissue in culture. To determine whether WNT11 could promote cardiac differentiation in tissue that was noncardiogenic, this molecule was ectopically expressed or added to mesoderm/endoderm explants obtained from stage 5 or 6 posterior tissue. Transfection of stage 5 posterior tissue with a WNT11 expression plasmid provoked the appearance of cardiomyocytes in 33% of the explants; half of which were contractile. Similarly transfected stage 6 posterior explants did not demonstrate cardiac differentiation. More dramatic results were obtained when noncardiogenic tissue was exposed to conditioned media containing soluble WNT11; as 63% and 33% of posterior stage 5- or stage 6-derived explants underwent cardiac differentiation. Together, these results indicate that WNT11 can promote cardiac development within noncardiac tissue. The expression of WNT11 in anterior mesoderm of early gastrula stage embryos suggests it may play a role in the formation of the vertebrate heart. Dev Dyn 1999;216:45-58.
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Glicoproteínas/fisiología , Corazón/embriología , Animales , Coturnix , Medios de Cultivo Condicionados , Regulación del Desarrollo de la Expresión Génica , Glicoproteínas/genética , Mesodermo/metabolismo , Miocardio/metabolismo , Sarcómeros/metabolismo , Transfección , Proteínas WntRESUMEN
The beginning of mesodermal development involves the aggregation of newly gastrulated cells into epithelial fields, as a prelude to organ formation. To analyze the molecular regulation of this initial patterning, we have focused on the Wnt family of secreted signaling proteins, molecules which have been shown to promote embryonic patterning by regulating cell-cell associations. In this study, we show that the Wnt-11 gene is expressed by newly gastrulated mesoderm cells within avian embryos. The expression pattern of Wnt-11 also suggests that it may be involved in formation of the cardiogenic fields and somites. Subsequently, we utilized the quail mesoderm cell line QCE-6 as a culture model for examining the influence of Wnt-11 on early mesoderm cell differentiation. This cell line has been shown to be representative of early nondifferentiated mesoderm cells and has the potential to differentiate into cardiomyocytes, endothelial or red blood cells. Similar to early mesoderm cells, QCE-6 cells express Wnt-11. We have engineered stable transfectants of these cells that produce either diminished or enhanced levels of Wnt-11 protein. Our studies show that Wnt-11 regulates cellular interactions of QCE-6 cells, as demonstrated by alterations in contact-inhibited growth, tight and gap junction formation and plakoglobin expression. Both the morphology and growth factor-induced differentiation of QCE-6 cells are regulated in a cooperative fashion by Wnt-11 and fibronectin. These results, described in detail below, demonstrate the uniqueness of QCE-6 cells as a culture system for analyzing Wnt activity. In particular, QCE-6 cells are the first cell line that has demonstrated: (1) Wnt-dependent differentiation; (2) concentration-variable responses to Wnt protein; and (3) altered cell phenotypes as a direct response to Wnt-5a class proteins (e.g. Wnt-4 and Wnt-11).
Asunto(s)
Gástrula/fisiología , Glicoproteínas/biosíntesis , Mesodermo/citología , Mesodermo/fisiología , Secuencia de Aminoácidos , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Pollos , Clonación Molecular , Embrión no Mamífero/citología , Embrión no Mamífero/fisiología , Fibronectinas/fisiología , Gástrula/citología , Regulación del Desarrollo de la Expresión Génica , Biblioteca de Genes , Glicoproteínas/química , Glicoproteínas/fisiología , Sustancias de Crecimiento/farmacología , Ratones , Datos de Secuencia Molecular , Codorniz , Ranidae , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Transducción de Señal , Transfección , Proteínas WntRESUMEN
The Wnt gene family consists of at least 15 structurally related genes that encode secreted extracellular signaling factors. Wnt proteins function in a range of critical developmental processes in both vertebrates and invertebrates and are implicated in regulation of cell growth and differentiation in certain adult mammalian tissues, including the mammary gland. We have isolated a number of WNT sequences from human genomic DNA, two of which, designated WNT14 and WNT15, represent novel members of the Wnt gene family. We also isolated WNT sequences from human mammary cDNA and present evidence that WNT13 is expressed in human breast tissue, in addition to those previously described. WNT14 and WNT15 appear to have originated from an ancestral branch of the Wnt gene family that also includes the Wnt9 sequences found in jawless and cartilaginous fishes. A Wnt14 cDNA was also isolated from chicken and a partial Wnt15 sequence from mouse. We show that human WNT14 maps to chromosome 1 and that WNT15 maps distal to BRCA1 on chromosome 17q21, where it lies within 125 kb of another WNT family member, WNT3.
Asunto(s)
Cromosomas Humanos Par 17 , Cromosomas Humanos Par 1 , Glicoproteínas , Proteínas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mama/metabolismo , Línea Celular , Cricetinae , ADN Complementario , Evolución Molecular , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , ARN , Homología de Secuencia de Aminoácido , Proteínas WntRESUMEN
The majority of congenital heart defects arise from abnormal development of valvuloseptal tissue. The primordia of the valve leaflets and membranous septa of the heart are the cardiac cushions. Remodeling of the cushions is associated with a transitional extracellular matrix that includes sulfated proteoglycans and the microfibrillar proteins fibulin and fibrillin. Cushion formation is restricted to the AV canal and ventricular outflow tract regions of the primary heart tube. The proper placement of the cushions may be the result of the development of the primary heart tube as a segmented organ, as well as the subsequent looping of the heart. Segmentation of the heart tube may be demonstrated by the alternating molecular expression pattern along the longitudinal axis. In support of this hypothesis is the restricted expression of BMP-4 and msx-2 to the AV canal and ventricular outflow tract. The importance of looping for cushion positioning may imply that the iv and inv genes and retinoic acid are important for the proper patterning of the heart. The cells of the cushions evolve from endocardial cells that undergo an epithelial-to-mesenchymal transformation. This developmental event is regulated by the myocardium and is probably due to the production of protein complexes, present within the cardiac jelly of the cushion-forming regions, that consist of fibronectin and the ES proteins. Both the cushion mesenchyme and its endocardial cell antecedents express JB3, an ECM protein. JB3 expression is also featured within the heart-forming fields of the primary mesoderm, from which the endocardial progenitors of the cushion cells originate.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Defectos de la Almohadilla Endocárdica/embriología , Endocardio/embriología , Regulación del Desarrollo de la Expresión Génica , Tabiques Cardíacos/embriología , Válvulas Cardíacas/embriología , Corazón/embriología , Morfogénesis , Animales , Diferenciación Celular , Línea Celular , Células Cultivadas , Coturnix , Peces , Atrios Cardíacos/embriología , Ventrículos Cardíacos/embriología , Técnicas In Vitro , Mesodermo/citología , Transcripción Genética , Transformación GenéticaRESUMEN
The Wnt gene family in vertebrates comprises at least 11 distinct genes but the only family member previously identified in Drosophila has been the segment polarity gene wingless (wg), the ortholog of vertebrate Wnt-1. In this report we describe the isolation of a novel Drosophila Wnt gene, Dwnt-5, which differs significantly from wg in both the pattern of its expression during embryogenesis and the predicted structure of its product. Dwnt-5 encodes a polypeptide of 112 kDa, which is more than twice as large as the products of previously known Wnt genes. The protein shares homology with other Wnt sequences in its carboxy-terminal half only and is most closely related to the products of vertebrate Wnt-5a and Wnt-5b. Dwnt-5 is expressed in a complex pattern during Drosophila embryogenesis. At the extended germ band stage, however, transcripts accumulate specifically in the nascent limb primordia of the head and thoracic segments. We show that this elevated expression depends on the activity of the homeobox gene Distal-less (Dll) and suggest that the Dwnt-5 gene may constitute a downstream target of Dll that acts in the specification of these primordia.
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Drosophila melanogaster/genética , Genes Homeobox , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Drosophila melanogaster/embriología , Extremidades/embriología , Regulación de la Expresión Génica , Datos de Secuencia MolecularRESUMEN
To evaluate the function of the murine ornithine decarboxylase (ODC) gene promoter, expression of chimeric ODC-chloramphenicol acetyltransferase (CAT) plasmids (pODCcat) containing 1,658 nt of the ODC promoter sequence and its various 5'-deletions was analyzed. In transient expression assays with NIH/3T3 mouse cells, pODCcat constructs exhibited fairly strong promoter activity yielding CAT values up to 40% of those obtained with the viral promoter RSV. Interestingly, 5'-deletions of the pODCcat constructs increased the promoter activity over that achieved using the entire 1.6-kb 5'-flanking region, with the highest activity being observed with about 750 nt of the ODC promoter. This finding suggests that the distal part of the promoter includes DNA elements which are involved in repressing its function. The promoter region could be deleted down to the proximal 97 nt and still be stimulated by cAMP to the same extent as the 1.6-kb promoter. DNase I footprinting and methylation interference studies showed that a specific protein binds to the region from -59 to -39, which encompasses a DNA motif resembling the consensus cyclic AMP response element (CRE). However, comparative gel retardation and Southwestern blotting experiments with the putative ODC-CRE and the somatostatin promoter CRE indicated that the 70-kDa protein interacting with the CRE-like element of the ODC promoter is different from the well-characterized nuclear CRE-binding protein CREB.
Asunto(s)
AMP Cíclico/fisiología , Proteínas de Unión al ADN/metabolismo , Ornitina Descarboxilasa/genética , Regiones Promotoras Genéticas , Animales , Secuencia de Bases , Unión Competitiva , Línea Celular , Clonación Molecular , ADN/genética , ADN/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Ratones , Datos de Secuencia Molecular , Ornitina Descarboxilasa/metabolismoRESUMEN
Ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) are two key enzymes in polyamine biosynthesis. Both the ODC and the AdoMetDC gene is regulated by androgens in accessory sex organs of mice and rats, whereas only the ODC gene is androgen-responsive in rodent kidney. Androgenic responses in murine and rat kidneys are, however, dissimilar in that the induction of ODC activity and ODC mRNA accumulation is transient in the rat but sustained in the murine renal cells. In addition, in situ hybridization experiments with single-stranded cRNA probes revealed that ODC gene expression occurs in different subpopulations of epithelial cells of the proximal tubules in mice and rats. ODC and AdoMetDC genes are androgen-regulated in the same cell types of the accessory sex organs, as judged by hybridization histochemistry. Sequencing of the promotor region of the murine ODC gene has indicated the presence of several DNA elements for binding of transcription factors/regulatory proteins, including a putative androgen-response element at about 900 nucleotides upstream of the transcription start site.