RESUMEN
Although the role of the b-cell lymphoma (Bcl)-2 family of apoptosis inhibitors is well documented in tumor cells and tissue morphogenesis, their role during the early development of vertebrates is unknown. Here, we characterize Nrz, a new Bcl-2-related inhibitor of apoptosis in zebrafish. Nrz is a mitochondrial protein, antagonizing the death-accelerator Bax. The nrz gene is mainly expressed during gastrulation and somitogenesis. The knockdown of nrz with antisense morpholinos leads to alterations of the somites, correlated with an increase in apoptosis. In addition, earlier during development, in the zebrafish gastrula, nrz knockdown results in an increase of snail-1 expression at the margin and frequent gastrulation arrest at the shield stage, independently of apoptosis. Together these data suggest that Nrz, in addition to its effect on apoptosis, contributes to cell movements during gastrulation by negatively regulating the expression of Snail-1, a transcription factor that controls cell adhesion.
Asunto(s)
Apoptosis/fisiología , Gástrula/fisiología , Proteínas Proto-Oncogénicas/fisiología , Somitos/fisiología , Proteínas de Pez Cebra/fisiología , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Western Blotting , Células COS , Chlorocebus aethiops , Clonación Molecular , Gástrula/citología , Gástrula/metabolismo , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Microscopía Confocal , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Factores de Transcripción de la Familia Snail , Somitos/citología , Somitos/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Pez Cebra/embriología , Pez Cebra/fisiología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
We reported previously that a 1.9-kb 5'-fragment from the human COL1A1 gene drove transcription of a promoterless human COL2A1 gene in tissues of transgenic mice that normally express the COL1A1 but not the COL2A1 gene. In the present study, we have established that the aberrant transcription of the COL2A1 gene did not produce any gross or microscopic phenotype, because the transcripts were not efficiently translated in cells that do not normally express the COL2A1 gene. In two lines of transgenic mice, the mRNA levels from the transgene were 30% to 45% of the mRNA for the proalpha1(I) chain of type I procollagen, the most abundant mRNA in the same tissues. Analysis of collagens extracted from skin of the transgenic mice indicated that triple-helical type II collagen, with the normal pattern of cyanogen bromide peptides, was synthesized from the transgene. However, the level of type II collagen in skin was less than 2% of the level of type I collagen. Hybridization in situ indicated the presence of mRNA for both COL2A1 and COL1A1 in the same cells. Immunofluorescence staining for type II collagen, however, was negative in the same tissues. The results, therefore, indicated that many mesenchymal cells in the transgenic mice had high steady-state levels of the homologous mRNAs for type I and type II procollagen, but only the mRNAs for type I procollagen were efficiently translated.
Asunto(s)
Colágeno/genética , Regulación hacia Abajo , Ratones Transgénicos , Biosíntesis de Proteínas , ARN Mensajero/biosíntesis , Animales , Células Cultivadas , Colágeno/biosíntesis , Colágeno/ultraestructura , Fibroblastos/citología , Técnica del Anticuerpo Fluorescente , Hibridación in Situ , Mandíbula/ultraestructura , Ratones , Fenotipo , Procolágeno/metabolismo , Regiones Promotoras Genéticas , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/biosíntesis , Piel/citología , Piel/metabolismo , Piel/ultraestructura , Tendones/ultraestructura , Diente/ultraestructura , Apófisis Xifoides/ultraestructuraAsunto(s)
ADN/genética , ADN/metabolismo , Hibridación in Situ/métodos , ARN/genética , ARN/metabolismo , Resinas Acrílicas , Animales , Autorradiografía , Compartimento Celular , Embrión de Pollo , Colágeno/genética , Fibronectinas/genética , Inmunohistoquímica , Microscopía Electrónica , Sondas Moleculares , Adhesión del Tejido , Fijación del TejidoRESUMEN
Detection of nucleic acid sequence at the ultrastructural level has allowed us to better understand the expression of genes in some fields of application in cell biology. In situ hybridization at the ultrastructural level can be carried out using three different methods: on vibratome sections before embedding in epoxy resin, on ultrathin frozen section, or on ultrathin section of tissue embedded in hydrophilic resin such as Lowicryl. Before starting the detection of nucleic acid sequences at the electron microscope level, the experimenter has to choose various parameters; the type of tissue fixation, the probe and its label, and the in situ hybridization method, depending on the sensitivity, the resolution and the ultrastructural preservation required. This review of technical aspects, by describing the different methods of ultrastructural in situ hybridization, will help the experimenter to optimize each step of the hybridization procedure.
Asunto(s)
Hibridación in Situ/métodos , Microscopía Electrónica/métodos , Animales , Encéfalo/metabolismo , Encéfalo/ultraestructura , Embrión de Pollo , Colágeno/genética , Femenino , Fibronectinas/genética , Secciones por Congelación , Expresión Génica , Hormona del Crecimiento/genética , Humanos , Sondas Moleculares , Oxitocina/genética , Hipófisis/metabolismo , Hipófisis/ultraestructura , ARN/genética , ARN/metabolismo , Ratas , Adhesión del Tejido , Fijación del TejidoRESUMEN
The ability of scale-forming cells to produce both type I and type V collagens was investigated by in situ hybridization at the light and electron microscope levels. Biochemical analyses reported that type I collagen, the predominant component, was associated with the minor type V collagen in the collagenous matrix of the teleost scales where, thin and thick collagen fibrils formed distinct layers. Thin collagen fibrils of the external layer were produced by the episquamal scleroblasts scattered on the outer scale surface, while thick collagen fibrils forming the compact basal plate were produced by the hyposquamal scleroblasts lining the inner surface of the scale. We demonstrated that episquamal and hyposquamal scleroblasts contained mRNAs for alpha1(I) and alpha1(V) collagens. Quantification by image analysis of the relative amount of alpha1(I) and alpha1(V) mRNAs in episquamal and hyposquamal scleroblasts suggests that the gene expression of type V collagen was proportionally higher in episquamal scleroblasts. These results support our hypothesis that the diameter of the thin fibrils of the external layer is regulated by the significant amount of type V collagen that interacts with type I collagen.
Asunto(s)
Cartílago/metabolismo , Animales , Células Cultivadas , Carpa Dorada , Hibridación in Situ , ARN Mensajero/biosíntesisRESUMEN
PURPOSE: To investigate the participation of interleukin-6 (IL-6) after photorefractive keratectomy (PRK) and its possible roles and sources in corneal wound healing. METHODS: IL-6, levels were measured in the tear fluids of patients before and after PRK and in conditioned media of human corneal epithelial cells and keratocytes. Its effects on total collagen and collagen-type synthesis by keratocytes were studied with a 3H-proline incorporation assay and Northern blot analysis. Zymography was used to evaluate the metalloproteinase content in the conditioned medium of IL-6-stimulated keratocytes. RESULTS: IL-6 is present in the tear fluid samples after photorefractive keratectomy, possibly synthesized by epithelial cells and keratocytes. CONCLUSIONS: IL-6 stimulates collagen synthesis in general and collagen type I in particular. Furthermore, it reduces the production of MMP-2, the latent form of the metalloproteinase, by cultured keratocytes. The results suggest that IL-6 might be regarded as a mediator involved in corneal healing after excimer laser.
Asunto(s)
Córnea/metabolismo , Interleucina-6/metabolismo , Queratectomía Fotorrefractiva , Lágrimas/metabolismo , Adulto , Northern Blotting , Técnicas de Cultivo de Célula , Colágeno/biosíntesis , Colágeno/genética , Córnea/citología , Córnea/efectos de los fármacos , Córnea/cirugía , Medios de Cultivo Condicionados , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Epitelio/cirugía , Femenino , Gelatinasas/biosíntesis , Humanos , Interleucina-6/farmacología , Láseres de Excímeros , Masculino , Metaloproteinasa 2 de la Matriz , Metaloendopeptidasas/biosíntesis , Miopía/cirugía , ARN Mensajero/biosíntesisRESUMEN
The skin is a tissue containing a large number of collagen types. Several collagens are restricted at the dermo-epidermal junction, contrarily to others present throughout the dermis. However, the distribution of the dermal collagen varies during embryonic development. In this contribution, we have been interested in the collagen types associated with the major collagenous components of the dermis, which are the collagen types I and III. Type V collagen, which is mixed with collagen types I and III to form heterotypic fibrils, has been studied during mouse embryo development. Transcripts of the alpha 1 (V) gene have been localized by in situ hybridization, on flattened cells of the stratum germinativum first, and then only on dermal cells. The expression of the gene decreases at birth, while the expression of the alpha 1(I) gene remains constant, with, however, a ring of high intensity around hair follicles. Other collagen types (VI, and the fibril-associated collagens XII and XIV) have been studied during calf embryonic development by immunofluorescence and ultrastructural immunogold detection. Type VI collagen appears homogeneously distributed throughout the dermis. Type XII collagen is first widely distributed and becomes restricted in the upper, papillary dermis after 6 months of gestation. Type XIV collagen, on the contrary, is first located as a delicate framework around hair follicles (at 19 weeks of gestation), and progressively invades the whole dermis where it appears abundant just before birth. The different functions of all these collagens are discussed in terms of dermis architecture, mechanical properties and physiology.
Asunto(s)
Colágeno/metabolismo , Colágeno/ultraestructura , Piel/crecimiento & desarrollo , Piel/metabolismo , Citoesqueleto de Actina/ultraestructura , Animales , Bovinos , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Ratones , Microscopía Electrónica , Microscopía Electrónica de Rastreo , Microscopía Inmunoelectrónica , Sondas ARN , Piel/ultraestructura , Distribución TisularRESUMEN
The expression of type I, II and III collagens genes was examined in human normal and hypochondrogenesis cartilage canals employing electrophoretic analysis, immunohistochemistry and in situ hybridization techniques. In normal cartilage, collagens type I and III were present in perichondrium, in the connective tissue surrounding the vessels of cartilage canals and in the dense fibrous tissue. However, types I and III procollagen mRNAs were detected only in fibroblasts of the perichondrium and of the canals, but not in the polymorphic cells. Type II collagen was present in the cartilage matrix and in the dense fibrous tissue, in good accordance with the localization of type II procollagen mRNAs detected in the chondrocytes and in the polymorphic cells. These data suggest that there are no transitional cells expressing type I, II and III collagen genes and that polymorphic cells are of chondrocytic origin. In the case of hypochondrogenesis, type II collagen was less abundant than in normal cartilage, whereas the corresponding mRNA level was equivalent. That suggests that a postranscriptional regulation of this protein is involved in the decrease of type II collagen production. Type I collagen, unexpectedly detected in the cartilage matrix, was synthesized by chondrocytes and polymorphic cells, suggesting a replacement of type II by type I collagen. The canal hypertrophy observed in this pathological case could thus be due to a modification in the regulation of the growth of cartilage canals caused by a defective cartilage matrix.
Asunto(s)
Enfermedades de los Cartílagos/genética , Cartílago/embriología , Cartílago/fisiología , Colágeno/genética , Cartílago/química , Enfermedades de los Cartílagos/metabolismo , Colágeno/análisis , Electroforesis , Epífisis/química , Epífisis/embriología , Expresión Génica , Humanos , Inmunohistoquímica , Hibridación in Situ , MutaciónRESUMEN
In situ hybridization for the detection of RNAm in ultrathin tissue sections embedded in lowicryl resin is reported. This method proved suitable for both tissues and cell cultures, using either complementary DNA probes or synthetic oligonucleotides, labeled with a radioisotope or biotin. This method seems to strike a satisfactory balance between preservation of cell ultrastructure and sensitivity.
Asunto(s)
Resinas Acrílicas , Hibridación in Situ/métodos , ARN Mensajero/análisis , Animales , Neoplasias de la Mama/genética , Receptores ErbB/genética , Femenino , Hormona del Crecimiento/genética , Humanos , Técnicas In Vitro , Masculino , Microscopía Electrónica , ARN Mensajero/ultraestructura , Ratas , Ratas WistarRESUMEN
In situ hybridization at the ultrastructural level can be carried out using three different methods: on vibratome sections before embedding in epoxy resin, on ultra-thin frozen sections, or on ultra-thin sections of tissues embedded in hydrophilic resin such as Lowicryl. With the purpose of comparing the sensitivity, resolution, and ultrastructural preservation of these three methods, we examined the expression of the growth hormone (GH) gene in anterior pituitary cells by in situ hybridization at the ultrastructural level, using a synthetic oligonucleotide complementary to the codons of the mRNA from Gln 45 to Ser 54 labeled at the 3' end of biotin-21dUTP. All these methods gave similar results: mRNA was located on the lamellar endoplasmic reticulum of somatotrophs. The pre-embedding method gave the best ultrastructural preservation, with low resolution with the enzymatic detection system and an intermediate sensitivity. A probe concentration of 10 pmol/ml was sufficient to obtain a signal. With this method gold particles could not be used without pre-treatment. The frozen section method gave the best sensitivity (a signal was observed with 4 pmol/ml of probe) but the lowest ultrastructural preservation. On ultra-thin Lowicryl sections, resolution was as high as with the frozen-section method, ultrastructural conservation was intermediate, and sensitivity was low. These results indicate that the last method seems to be a good compromise between sensitivity and ultrastructural preservation.
Asunto(s)
Hormona del Crecimiento/análisis , Microscopía Electrónica/métodos , Adenohipófisis/química , ARN Mensajero/análisis , Animales , Secciones por Congelación , Masculino , Hibridación de Ácido Nucleico , Adenohipófisis/ultraestructura , Ratas , Ratas Endogámicas , Adhesión del Tejido/métodosRESUMEN
The expression of mRNAs for collagen types I, II, III and for aggrecan core protein was studied in developing human femoral cartilage by in situ hybridization, with special attention given to the cartilage covered by the perichondrium and to the articular surface. In parallel, the synthesis of the related proteins was monitored by immunohistochemistry. The cells metabolically active for type I and type III collagen expression were identified by hybridization using [32P]-labeled cDNA clones coding for human alpha 1(I) and alpha 1(III), respectively. Type II collagen and core protein mRNAs were detected by hybridization with specific [32P]-labeled oligonucleotide probes. In the femoral heads of one 22-week old fetus and of one newborn, our in situ hybridization and immunohistochemical analysis revealed that chondrocytes located immediately subjacent to the perichondrium produced collagen types I, II, III as well as aggrecan; whereas only type II collagen and aggrecan gene expression was detected deeper in the cartilage covered by the perichondrium. This observation supports the hypothesis that the inner cell layers of perichondrium are chondrogenic, with a transient state where cells express all the markers studied here. At the articular surface different patterns of expression were observed at the two developmental stages. After 22 weeks of fetal development only collagen types I and III were expressed by the surface zone cells while in the newborn cartilage, these cells expressed all the molecules studied (collagen types I, II, III and cartilage proteoglycan). At both ages the underlying cartilage cells expressed only the cartilage-specific molecules (type II collagen and aggrecan). Thus a progressive transformation of cartilaginous matrix occurs with time from the deep cartilage up to the surface by addition of new components, i.e. aggrecan and type II collagen. These results supplemented by an immunofluorescence analysis on 20-, 26- and 38-week old fetal femoral heads suggest that expression of collagen and aggrecan in the cartilage covered by the perichondrium and in the cartilage at the articular surface are subject to different regulatory mechanisms during development. Furthermore, the appearance of hybridizable core protein and type II collagen mRNAs at the articular surface, closely followed by the appearance of the proteins for which they code, indicates that core protein and type II collagen expression is regulated primarily at the transcriptional level in this region. Finally, the similar topography observed for the expression of these two proteins suggests that the genes for these two major constituents of cartilage matrix are coordinately regulated during growth of articular cartilage.
Asunto(s)
Cartílago Articular/metabolismo , Colágeno/biosíntesis , Proteínas de la Matriz Extracelular , Expresión Génica , Proteoglicanos/biosíntesis , Agrecanos , Secuencia de Bases , Cartílago Articular/embriología , Colágeno/clasificación , Colágeno/genética , Fémur/embriología , Edad Gestacional , Humanos , Recién Nacido , Lectinas Tipo C , Datos de Secuencia Molecular , Familia de Multigenes , Hibridación de Ácido Nucleico , Proteoglicanos/genética , ARN Mensajero/biosíntesisRESUMEN
Two previously described sponge cDNAs, EmC4 and C23, respectively encoding a short chain collagen and a fibrillar collagen, were used to characterize collagen gene families in a freshwater sponge. EmC4 detected several clones when used to screen a cDNA library. Two overlapping clones, EmC13 1 and 2, were sequenced and appeared highly homologous to EmC4. Contrarily to C23, EmC4 hybridized with 10-12 fragments of genomic DNA digested with restriction endonucleases and detected 10 times more positive clones than C23 when used to screen a genomic library. The genomic clone G41 contained two closely related genes, COLNF13, corresponding to EmC13 and COLNF6. Partial characterization of COLNF13 revealed two partial exons and four complete exons of 153, 219, 207, and 144 base pairs, with split glycine codons at their boundaries. The deduced encoded protein is a short chain collagen containing two uninterrupted collagenous domains of 66 and 171 amino acids and non-collagenous domains. A characterized 207-base pair exon of COLNF6 is 77% identical with the comparable COLNF13 exon. In situ hybridization using EmC4 cDNA and electron microscopy suggested that the cells expressing these genes were secreting spongin, a non-fibrillar, surface collagen of these sponges.
Asunto(s)
Colágeno/genética , Familia de Multigenes , Poríferos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Clonación Molecular , ADN/genética , ADN/aislamiento & purificación , Biblioteca Genómica , Microscopía Electrónica , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Poríferos/ultraestructura , Mapeo RestrictivoRESUMEN
The EGF receptor (EGF-R), a 170 KD transmembrane glycoprotein, is found at a high level in the BT20 human mammary carcinoma cell line (1 +/- 0.4 x 10(6) sites per cell). In this study, we examined the expression of the EGF-R gene in BT20 cell line by in situ hybridization at the light and electron microscopic level using a human cDNA, corresponding to EGF-R transmembrane and protein kinase domains, labeled with [3H]-, [35S]-, or [32P]-d-ATP. Two treatments were tested to embed cells in Lowicryl resin: the first used fixation and dehydration by progressive lowering of temperature, the second quick freezing and cryosubstitution. The best ultrastructural preservation was obtained with the second procedure without modification of the hybridization signal. EGF-R mRNA was observed principally at the cytoplasmic level, on organelles involved in the protein synthesis process. Labeling was also located on the microvilli which extend into the intercellular space, suggesting that some mRNA would be located in sites where EGF-R is utilized. Some mRNA was observed in the nucleus. This study demonstrates that post-embedding in situ hybridization, after quick freezing and cryosubstitution, is a powerful EM in situ hybridization procedure to study the expression of the EGF-R gene.
Asunto(s)
Neoplasias de la Mama/ultraestructura , Receptores ErbB/genética , Expresión Génica , Hibridación de Ácido Nucleico , ARN Mensajero/análisis , Citoplasma/química , Sondas de ADN , Retículo Endoplásmico/química , Humanos , Microscopía Electrónica , Microvellosidades/química , Membrana Nuclear/química , Ribonucleasa Pancreática , Radioisótopos de Azufre , Tritio , Células Tumorales CultivadasRESUMEN
Human skeletal tissues fixed in Bouin's solution and embedded in paraffin were studied in order to identify cells responsible for production of types I and II collagens by in situ hybridization. The probes used were a double stranded cDNA fragment (type I collagen) and a synthetic oligonucleotide (type II collagen). The specificity of these probes labeled with 32P was proven in hybridizations to sections of human fetal femoral heads: fibroblasts and chondrocytes, known to produce respectively type I and type II collagen were only recognized by the corresponding probes. These results suggest that paraffin blocks available in pathology departments could be reexamined by in situ hybridization. Furthermore, the use of synthetic oligonucleotides without cloning the gene of interest could increase the usefulness of the technique in studies on human cartilage diseases and possibly on other diseases.
Asunto(s)
Acetatos , Ácido Acético , Fijadores , Formaldehído , Hibridación de Ácido Nucleico , Parafina , Picratos , ARN Mensajero/análisis , Autorradiografía , Secuencia de Bases , Cartílago/química , Cartílago/embriología , Sondas de ADN , Cabeza Femoral/química , Cabeza Femoral/embriología , Fibroblastos/química , Técnicas Genéticas , Humanos , Datos de Secuencia MolecularRESUMEN
We have studied the expression of the fibronectin gene in 7 day-old chick embryo (stage 32) by in situ hybridization at the light and electron microscope levels, using a 397 base-pairs chicken cDNA, labeled by radioisotope or biotin-11dUTP. Cryostat sections of whole chick embryos displayed a selective label on the upper layer of the dermis, fibrous sclera and mesenchymal cells but not on cartilagenous sclera cells. These results show that the expression of the fibronectin gene varies in relation to the morphogenetic events. Hybridization at the ultrastructural level on thin sections of sclera embedded in Lowicryl K4M showed a selective labelling on various cell compartments. Biotin-11dUTP and radiolabeled probes were compared. The labeling was found precisely on the membrane of the rough endoplasmic reticulum and on the nuclear envelope. A few silver grains were located on the nucleus and in the perinucleolar region. This study shows that the postembedding in situ hybridization is a powerful procedure to study the expression of the extracellular protein genes and gives further information on the localization of mRNA.
Asunto(s)
Fibronectinas/análisis , Animales , Biotina , Embrión de Pollo , Sondas de ADN , Epidermis/química , Epidermis/ultraestructura , Fibronectinas/genética , Microscopía Electrónica , Hibridación de Ácido Nucleico , ARN Mensajero/metabolismo , Esclerótica/química , Esclerótica/ultraestructura , Radioisótopos de AzufreRESUMEN
The distribution of sites of type I collagen gene expression was studied in frozen sections of skin of 4 and 9 month-old calf fetuses by in situ hybridization using a human pro-alpha 1 type I collagen cDNA. The labelling varied with the different layers of the dermis and with the developmental stage considered. In the 4 month old fetus skin, the label appeared concentrated in the upper layer of the dermis at the lewel of the hair follicles. In the 9 month-old fetus skin, the difference of labelling between upper papillary dermis and lower dermis was less marked. Comparatively the distribution of the extracellular type I collagen was determined by indirect immunofluorescence. This collagen appeared present throughout the whole dermis with slight variations at 4 months, where there was less extracellular collagen near the hair bulbs. These results are in agreement with the idea that the collagen synthesis follows cutaneous differentiation. In addition, they support the hypothesis that collagen is deposited once morphogenetic events have occurred and plays thus a stabilizing role in formation of cutaneous appendages.
Asunto(s)
Colágeno/metabolismo , Desarrollo Embrionario y Fetal , Regulación de la Expresión Génica , ARN Mensajero/metabolismo , Piel/metabolismo , Animales , Bovinos , Inmunohistoquímica , Hibridación de Ácido Nucleico , Piel/citología , Piel/embriologíaRESUMEN
To examine the mechanism of viral tropism in vivo, we injected RAV(1) avian retrovirus into 1-day-old chicken embryos. After 8 days, the majority of the embryos became infected. In situ hybridization to whole embryo sections revealed high levels of intracellular viral RNA, apparently restricted to skeletal muscle cells. The most likely interpretation is that expression of this virus is regulated at the transcriptional level by one or more tissue-specific endogenous factors.
Asunto(s)
Virus de la Leucosis Aviar/aislamiento & purificación , Embrión de Pollo/microbiología , Regulación de la Expresión Génica , Animales , Virus de la Leucosis Aviar/fisiología , Músculos/análisis , Hibridación de Ácido Nucleico , Especificidad de Órganos , ARN Viral/análisis , Transcripción GenéticaRESUMEN
We analyzed the expression of the c-erbA proto-oncogene in different tissues of chicken embryos. c-erbA transcripts were found at low levels in the lung, kidney, liver, and heart and in high amounts in embryonic blood cells. Nuclease mapping assays proved that these transcripts were true c-erbA transcripts. In situ hybridization on fractionated embryonic blood cells showed that c-erbA transcripts were predominantly found in erythroblasts, particularly during the final step of differentiation. Life span analysis of c-erbA mRNAs revealed their relative instability, demonstrating that the high level of c-erbA transcripts in embryonic erythroblasts was not the result of passive accumulation. These results suggest that the c-erbA genes play some role in erythrocyte differentiation.
Asunto(s)
Eritrocitos/metabolismo , Regulación de la Expresión Génica , Proto-Oncogenes , Animales , Embrión de Pollo , Eritrocitos/citología , Eritropoyesis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción GenéticaRESUMEN
In vitro metabolism of androstenedione in gonads of juvenile and adult Helix aspersa has been investigated. The conversion of [3H]androstenedione into testosterone, 5 alpha-dihydrotestosterone, androsterone, and estriol was demonstrated. In juvenile animals testosterone (59.8%) is the major metabolite whereas in adult animals androsterone (18.8%) is. The following endogenous steroids have been identified by gas chromatography-mass spectrometry in adult gonads: androsterone, dehydroepiandrosterone, androstenedione, 3 alpha-androstanediol, estrone, estradiol-17 beta, and estriol. The levels of testosterone, 5 alpha-dihydrotestosterone, androstenedione, and progesterone have been measured by RIAs in gonads and hemolymph. Their levels vary with the physiological stage: the gonadal and circulating levels of testosterone decrease with the sexual maturation whereas the 5 alpha-dihydrotestosterone increases. These differences observed in metabolism and in level of steroids between the juvenile and the adult snails allow us to suppose that these steroids have a biological role.