RESUMEN
We have used P19 embryonal carcinoma cells as in vitro model for early neurogenesis to study ionotropic P2X and metabotropic P2Y receptor-induced Ca(2+) transients and their participation in induction of proliferation and differentiation. In embryonic P19 cells, P2Y(1), P2Y(2) and P2X(4) receptors or P2X-heteromultimers with similar P2X(4) pharmacology were responsible for ATP and ATP analogue-induced Ca(2+) transients. In neuronal-differentiated cells, P2Y(2,) P2Y(6), P2X(2) and possibly P2X(2)/P2X(6) heteromeric receptors were the major mediators of the elevations in intracellular free calcium concentration [Ca(2+)](i). We have collected evidence for the involvement of metabotropic purinergic receptors in proliferation induction of undifferentiated and neural progenitor cells by using a BrdU-incorporation assay. ATP-, UTP-, ADP-, 2-MeS-ATP- and ADP-betaS-induced proliferation in P19 cells was mediated by P2Y(1) and P2Y(2) receptors as judged from pharmacological profiles of receptor responses. ATP-provoked acceleration of neuronal differentiation, determined by analysis of nestin and neuron-specific enolase gene and protein expression, also resulted from P2Y(1) and P2Y(2) receptor activation. Proliferation- and differentiation-induction involved the activation of inositol-trisphosphate sensitive intracellular Ca(2+) stores.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Sistema Nervioso/embriología , Neurogénesis/fisiología , Neuronas/metabolismo , Receptores Purinérgicos/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Calcio/metabolismo , Señalización del Calcio/fisiología , Línea Celular Tumoral , Células Madre de Carcinoma Embrionario , Inducción Embrionaria/efectos de los fármacos , Inducción Embrionaria/fisiología , Humanos , Fosfatos de Inositol/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Sistema Nervioso/citología , Nestina , Neurogénesis/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Fosfopiruvato Hidratasa/metabolismo , Receptores Purinérgicos/efectos de los fármacos , Receptores Purinérgicos P2/efectos de los fármacos , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X2 , Receptores Purinérgicos P2Y1 , Receptores Purinérgicos P2Y2 , Células Madre/efectos de los fármacos , Células Madre/metabolismoRESUMEN
As an important step in the nuclear transfer (NT) procedure, we evaluated the effect of three different treatments for oocyte activation on the in vitro and in vivo developmental capacity of bovine reconstructed embryos: (1) strontium, which has been successfully used in mice but not yet tested in cattle; (2) ionomycin and 6-dimethylaminopurine (6-DMAP), a standard treatment used in cattle; (3) ionomycin and strontium, in place of 6-DMAP. As regards NT blastocyst development, no difference was observed when strontium (20.1%) or ionomycin/6-DMAP (14.4%) were used. However, when 6-DMAP was substituted by strontium (3), the blastocyst rate (34.8%) was superior to that in the other activation groups (p < 0.05). Results of in vivo development showed the possibility of pregnancies when NT embryos activated in strontium were transferred to recipient cows (16.6%). A live female calf was obtained when ionomycin/strontium were used, but it died 30 days after birth. Our findings show that strontium can be used as an activation agent in bovine cloning procedures and that activation with a combination of strontium and ionomycin increased the in vitro developmental capacity of reconstructed embryos. This is the first report of a calf produced by adult somatic cell NT in Latin America.
Asunto(s)
Inducción Embrionaria/efectos de los fármacos , Células Híbridas , Técnicas de Transferencia Nuclear , Oocitos/efectos de los fármacos , Estroncio/farmacología , Adenina/análogos & derivados , Adenina/farmacología , Animales , Bovinos , Clonación de Organismos , Femenino , Fertilización In Vitro , Ionomicina/farmacología , Ionóforos/farmacología , Repeticiones de Microsatélite , Partenogénesis , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas , Estimulación QuímicaRESUMEN
The induction and improvement of in vitro rhizogenesis of microshoots of Prosopis chilensis (Mol.) Stuntz and Nothofagus alpina (Poep. et Endl. Oerst.) were compared using Agrobacterium rhizogenes (Ar) versus indole-3-butyric acid (IBA) in the culture media. Microshoots of P. chilensis (1-2 cm length), coming from in vitro grown seedlings, were cultivated in a modified Broadleaved Tree Medium (BTMm) containing half salt concentration of macronutrients and 0.05 mg x L(-1) benzilaminopurine (BAP). After 30 days, microshoots with 2-4 leaves were selected and cultured in BTMm-agar in presence or abscense of Ar and in combination with IBA. For N. alpina, the apical shoots with the first 2 true leaves, from 5 weeks old seedlings, were cultured in the abovementioned medium, but with 0.15 mg x L(-1) of BAP. After 2 months, microshoots with 2-3 leaves were selected and cultured in BTMm-agar, supplemented with 5 mg x L(-1) IBA or in liquid BTMm on perlite and, in the presence or absence of A. rhizogenes (Ar) and in combination with 3 mg x L(-1) IBA. Rooting in P. chilensis reached 100.0% when Ar infection was produced in the presence of IBA, increasing both, the number and dry weight of roots. In N. alpina, 90.0% of rooting efficiency was obtained when Ar infection was produced in liquid culture and in the absence of auxin. (AU)
Asunto(s)
Estudio Comparativo , RESEARCH SUPPORT, NON-U.S. GOVT , Ácidos Indolacéticos/farmacología , Inducción Embrionaria/efectos de los fármacos , Raíces de Plantas/crecimiento & desarrollo , Prosopis/crecimiento & desarrollo , Rhizobium/fisiología , Árboles/crecimiento & desarrollo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Medios de Cultivo/química , Medios de Cultivo/farmacología , Fenómenos Fisiológicos de las Plantas/efectos de los fármacos , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/microbiología , Brotes de la Planta/efectos de los fármacos , Brotes de la Planta/crecimiento & desarrollo , Brotes de la Planta/microbiología , Prosopis/efectos de los fármacos , Prosopis/microbiología , Árboles/efectos de los fármacos , Árboles/microbiologíaRESUMEN
The induction and improvement of in vitro rhizogenesis of microshoots of Prosopis chilensis (Mol.) Stuntz and Nothofagus alpina (Poep. et Endl. Oerst.) were compared using Agrobacterium rhizogenes (Ar) versus indole-3-butyric acid (IBA) in the culture media. Microshoots of P. chilensis (1-2 cm length), coming from in vitro grown seedlings, were cultivated in a modified Broadleaved Tree Medium (BTMm) containing half salt concentration of macronutrients and 0.05 mg x L(-1) benzilaminopurine (BAP). After 30 days, microshoots with 2-4 leaves were selected and cultured in BTMm-agar in presence or abscense of Ar and in combination with IBA. For N. alpina, the apical shoots with the first 2 true leaves, from 5 weeks old seedlings, were cultured in the abovementioned medium, but with 0.15 mg x L(-1) of BAP. After 2 months, microshoots with 2-3 leaves were selected and cultured in BTMm-agar, supplemented with 5 mg x L(-1) IBA or in liquid BTMm on perlite and, in the presence or absence of A. rhizogenes (Ar) and in combination with 3 mg x L(-1) IBA. Rooting in P. chilensis reached 100.0% when Ar infection was produced in the presence of IBA, increasing both, the number and dry weight of roots. In N. alpina, 90.0% of rooting efficiency was obtained when Ar infection was produced in liquid culture and in the absence of auxin.
Asunto(s)
Árboles/crecimiento & desarrollo , Ácidos Indolacéticos , Inducción Embrionaria/efectos de los fármacos , Prosopis/crecimiento & desarrollo , Raíces de Plantas/crecimiento & desarrollo , Rhizobium/fisiología , Árboles/efectos de los fármacos , Árboles/microbiología , Brotes de la Planta/efectos de los fármacos , Brotes de la Planta/crecimiento & desarrollo , Brotes de la Planta/microbiología , Células Cultivadas , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Fenómenos Fisiológicos de las Plantas/efectos de los fármacos , Medios de Cultivo/química , Medios de Cultivo/farmacología , Prosopis/efectos de los fármacos , Prosopis/microbiología , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/microbiologíaRESUMEN
The induction and improvement of in vitro rhizogenesis of microshoots of Prosopis chilensis (Mol.) Stuntz and Nothofagus alpina (Poep. et Endl. Oerst.) were compared using Agrobacterium rhizogenes (Ar) versus indole-3-butyric acid (IBA) in the culture media. Microshoots of P. chilensis (1-2 cm length), coming from in vitro grown seedlings, were cultivated in a modified Broadleaved Tree Medium (BTMm) containing half salt concentration of macronutrients and 0.05 mg x L(-1) benzilaminopurine (BAP). After 30 days, microshoots with 2-4 leaves were selected and cultured in BTMm-agar in presence or abscense of Ar and in combination with IBA. For N. alpina, the apical shoots with the first 2 true leaves, from 5 weeks old seedlings, were cultured in the abovementioned medium, but with 0.15 mg x L(-1) of BAP. After 2 months, microshoots with 2-3 leaves were selected and cultured in BTMm-agar, supplemented with 5 mg x L(-1) IBA or in liquid BTMm on perlite and, in the presence or absence of A. rhizogenes (Ar) and in combination with 3 mg x L(-1) IBA. Rooting in P. chilensis reached 100.0% when Ar infection was produced in the presence of IBA, increasing both, the number and dry weight of roots. In N. alpina, 90.0% of rooting efficiency was obtained when Ar infection was produced in liquid culture and in the absence of auxin.
Asunto(s)
Inducción Embrionaria/efectos de los fármacos , Ácidos Indolacéticos/farmacología , Raíces de Plantas/crecimiento & desarrollo , Prosopis/crecimiento & desarrollo , Rhizobium/fisiología , Árboles/crecimiento & desarrollo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Medios de Cultivo/química , Medios de Cultivo/farmacología , Fenómenos Fisiológicos de las Plantas/efectos de los fármacos , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/microbiología , Brotes de la Planta/efectos de los fármacos , Brotes de la Planta/crecimiento & desarrollo , Brotes de la Planta/microbiología , Prosopis/efectos de los fármacos , Prosopis/microbiología , Árboles/efectos de los fármacos , Árboles/microbiologíaRESUMEN
The neural crest is a unique cell population induced at the lateral border of the neural plate. Neural crest is not produced at the anterior border of the neural plate, which is fated to become forebrain. Here, the roles of BMPs, FGFs, Wnts, and retinoic acid signaling in neural crest induction were analyzed by using an assay developed for investigating the posteriorization of the neural plate. Using specific markers for the anterior neural plate border and the neural crest, the posterior end of early neurula embryos was shown to be able to transform the anterior neural plate border into neural crest cells. In addition, tissue expressing anterior neural plate markers, induced by an intermediate level of BMP activity, was transformed into neural crest by posteriorizing signals. This transformation was mimicked by bFGF, Wnt-8, or retinoic acid treatment and was also inhibited by expression of the dominant negative forms of the FGF receptor, the retinoic acid receptor, and Wnt signaling molecules. The transformation of the anterior neural plate border into neural crest cells was also achieved in whole embryos, by retinoic acid treatment or by use of a constitutively active form of the retinoic acid receptor. By analyzing the expression of mesodermal markers and various graft experiments, the expression of the mutant retinoic acid receptor was shown to directly affect the ectoderm. We thereby propose a two-step model for neural crest induction. Initially, BMP levels intermediate to those required for neural plate and epidermal specification induce neural folds with an anterior character along the entire neural plate border. Subsequently, the most posterior region of this anterior neural plate border is transformed into the neural crest by the posteriorizing activity of FGFs, Wnts, and retinoic acid signals. We discuss a unifying model where lateralizing and posteriorizing signals are presented as two stages of the same inductive process required for neural crest induction.
Asunto(s)
Tipificación del Cuerpo/fisiología , Inducción Embrionaria/fisiología , Factores de Crecimiento de Fibroblastos/fisiología , Cresta Neural/fisiología , Proteínas Proto-Oncogénicas/fisiología , Receptores de Factores de Crecimiento , Tretinoina/fisiología , Proteínas de Pez Cebra , Animales , Biomarcadores , Tipificación del Cuerpo/efectos de los fármacos , Tipificación del Cuerpo/genética , Receptores de Proteínas Morfogenéticas Óseas , Proteínas Morfogenéticas Óseas/fisiología , Linaje de la Célula , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/fisiología , Inducción Embrionaria/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Gástrula/efectos de los fármacos , Gástrula/fisiología , Regulación del Desarrollo de la Expresión Génica , Microinyecciones , Cresta Neural/citología , Prosencéfalo/embriología , ARN Mensajero/administración & dosificación , ARN Mensajero/genética , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/fisiología , Receptores de Factores de Crecimiento de Fibroblastos/efectos de los fármacos , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento de Fibroblastos/fisiología , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/fisiología , Receptor alfa de Ácido Retinoico , Tretinoina/farmacología , Proteínas Wnt , Proteínas de Xenopus , Xenopus laevis/embriología , Xenopus laevis/genéticaRESUMEN
Mesoderm induction requires interaction between cells of the animal and vegetal hemispheres of the embryo. Several molecules have been proposed as candidates for mesoderm-inducing signals, with activin a particularly strong candidate. However, it has not been possible to inhibit mesoderm formation in vivo by specifically blocking activin action. Follistatin is able to inhibit the action of activin but not that of the mature region of Vg1, a member of the transforming growth factor beta family. Follistatin therefore provides a useful tool for distinguishing between signalling by these two factors. We have overexpressed Xenopus follistatin mRNA and analysed the expression of several mesodermal markers. Our results show an inhibition of mesodermal formation by follistatin in a concentration-dependent manner, showing the requirement of activin for mesodermal induction.
Asunto(s)
Proteínas Fetales , Glicoproteínas/farmacología , Inhibidores de Crecimiento/farmacología , Proteínas de Homeodominio , Mesodermo/efectos de los fármacos , Proteínas Represoras , Proteínas de Dominio T Box , Proteínas de Pez Cebra , Animales , Técnicas de Cultivo , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Inducción Embrionaria/efectos de los fármacos , Folistatina , Marcadores Genéticos , Glicoproteínas/genética , Proteína Goosecoide , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , ARN Mensajero , Proteínas Recombinantes/farmacología , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Proteínas Wnt , XenopusRESUMEN
The processes of cellular migration, cellular differentiation and cellular multiplication are studied, since these are the basic developmental processes upon which teratogenic agents act resulting in congenital malformations. We also carefully analyze the interactions between teratogen-embryo in order to establish adequate parameters for analysis of environmental teratogens, as well as experimental teratogenesis and epidemiology. Information on the pathogenesis of congenital malformations obtained from experimental teratology in an adequate biological model, can be extrapolated to the human. The etiology of congenital malformations resulting from environmental teratogens can only be elucidated through epidemiology, since there is species specificity. Such a study must fulfill the following prerequisites: diagnosis of the congenital malformation, ruling out genetic factors in the family tree and determination of the exact time of exposure to the possible teratogen during the pregnancy.
Asunto(s)
Anomalías Inducidas por Medicamentos/etiología , Contaminantes Ambientales/efectos adversos , Teratógenos , Anomalías Inducidas por Medicamentos/embriología , Anomalías Inducidas por Medicamentos/epidemiología , Anomalías Inducidas por Medicamentos/genética , Animales , Movimiento Celular/efectos de los fármacos , Inducción Embrionaria/efectos de los fármacos , Edad Gestacional , Cardiopatías Congénitas/inducido químicamente , Humanos , Modelos Biológicos , Morfogénesis/efectos de los fármacos , Proyectos de Investigación , Teratógenos/farmacologíaRESUMEN
We have studied the localization of the proteins of Xeb1 and Xeb2, two homeobox (hbx)-containing genes that are expressed during the early development of Xenopus laevis. Both proteins are expressed in juxtaposed and partially overlapping domains along the antero-posterior axis of Xenopus laevis embryos, with clearly defined anterior boundaries. Xeb2 is predominantly expressed in the caudal region of the hindbrain, whereas the Xeb1 protein is located in the most rostral region of the spinal cord. Furthermore, both proteins are expressed in single cells dispersed in the lateral flanks of the embryo in positions that correlate with the expression domains in the neural tube. We suggest that these cells are migratory neural crest cells that have acquired positional information in the neural tube prior to migration. The Xeb2 protein was also detected in the most posterior branchial arches and the pronephros. In stage 45 embryos, nuclei of the IX-X cranial ganglia, the lung buds and cells spreading into the forelimb rudiment express the Xeb2 antigen. The Xeb1 protein was also detected in the lung buds and the forelimb rudiment. To examine the effect of retinoic acid on expression, gastrula embryos were treated with all-trans retinoic acid (RA). Increasing concentrations of RA caused progressive truncation of anterior structures. The most severely affected embryos lacked eyes, nasal pits, forebrain, midbrain and otic vesicles, and the anterior boundary of the hindbrain seemed to be displaced rostrally. This alteration correlates with a progressive displacement of the anterior boundary of the expression domain of Xeb2. On the other hand, 10(-6) M RA induces an ectopic site of Xeb1 expression at the anterior end of the central nervous system, located just anterior to the extended domain of Xeb2 whereas expression in the spinal cord remains unaffected.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Genes Homeobox , Tretinoina/farmacología , Animales , Inducción Embrionaria/efectos de los fármacos , Inmunohistoquímica , Proteínas/análisis , Xenopus laevis/embriologíaRESUMEN
El Li+ tiene la capacidad, en embriones sanos, de inducir la formación supernumeraria de estructuras cefálicas y de revertir las deficiencias del eje neural que se evidencian en embriones expuestos a la luz ultravioleta. Por otra parte, la hidrólisis de los fosfoinositodos de la membrana se ha implicado en los aumentos de Ca2 que proceden a los eventos que se suceden durante la fertilización y el desarrollo embrionario y el Li+ impide que los niveles de fosfoinositol (4,5), bifosfato retornen a sus niveles normales. La conclusión a la cual se debe llegar es que el Li+ altera la reespecificación dorsoanterior de los embriones y que esto debe depender de su capacidad de interferir con el ciclo de los fosfoinositidos. Estas observaciones tienen relevancia particular puesto que los efectos específicos del Li+ sobre el ciclo de los fosfoinositidos permiten suponer que alguno de los intermediarios de este ciclo debe jugar un papel importante en la diferenciación y especificación dorsoanterior del embrión. Es preocupante el hecho de que estos efectos pueden obtenerse utilizando concentraciones de Li+ similares a las usadas terapéuticamente en el manejo de las enfermedades maniacodepresivas
Asunto(s)
Inducción Embrionaria/efectos de los fármacos , Litio/metabolismo , Fosfatidilinositoles/metabolismoRESUMEN
Presumptive ectoderm from young gastrulae of the Pleurodele and the Axolotl was treated with concanavalin A(5-25 micrograms/ml). Then, it was combined, in a sandwich, with the dorsal lip of the blastopore and cultured in vitro for 5-7 days. The results of the experiments show that the treated ectoderm becomes more fragile than untreated control ectoderm. Nevertheless, the neural induction and the histological differentiation of the neural tissue appeared in a rather normal fashion.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Concanavalina A/farmacología , Inducción Embrionaria/efectos de los fármacos , Gástrula/efectos de los fármacos , Sistema Nervioso/embriología , Ambystoma mexicanum , Animales , Ectodermo/efectos de los fármacos , PleurodelesRESUMEN
The dependence of the differentiation-pattern of competent amphibian ectoderm on the proportion of inducing and induced material was studied. To do so different masses of LiCl-treated tissue were combined with a constant mass of untreated material. LiCl-treated isolates corresponding in size with the treated portions in the combinations served as controls. The experiments seem to show at least three factors responsible for the differentiation-pattern of the combinations: the number of inducing cells, the number of cells to be induced, and the competence of the ectoderm, which depends on the stage of development.