RESUMEN
A major event affecting the eye during amphibian metamorphosis is an asymmetrical growth of the ventrotemporal portion of the retina compared with its dorsonasal counterpart. This event is due to an increased proliferation of the precursors of the ventral ciliary marginal zone (CMZ). Here, we analyze the expression patterns of several key homeobox genes implicated in eye development (Xrx1, Xvax2, Xsix3, Xpax6, Xchx10, Xotx2) to understand whether they are active at the time in which the metamorphic changes of the retina occur. We also analyze their expression patterns in the ventral and dorsal CMZ and compare them with bromodeoxyuridine incorporation in the CMZ. Our results suggest that the metamorphic CMZ maintains the functional subdivisions described during embryonic development. Moreover, we find that genes involved in proliferation and cell type determination of the embryonic retina are actively transcribed in the proliferating CMZ, thus indicating a potential regulatory role for these genes in the metamorphic retina.
Asunto(s)
Ojo/crecimiento & desarrollo , Proteínas de Homeodominio/genética , Metamorfosis Biológica/genética , Xenopus laevis/crecimiento & desarrollo , Xenopus laevis/genética , Animales , Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Homeodominio/metabolismo , Retina/crecimiento & desarrollo , Retina/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMEN
The PC3 gene is a marker of dividing neuroepithelial (NE) cells. We transduced single cortical precursors of the ventricular zone (VZ) with a PC3-carrying retroviral vector at E16 stage, and analysed the effects of transgene expression on their progeny in 3-week-old animals. Unlike control-transduced cells, all viable PC3-transduced cells remained close to the ventricle and displayed a round-shaped, undifferentiated morphology.
Asunto(s)
Ácido Aspártico Endopeptidasas/genética , Corteza Cerebral/citología , Corteza Cerebral/embriología , Regulación del Desarrollo de la Expresión Génica , Regulación Viral de la Expresión Génica , Animales , Diferenciación Celular/fisiología , Supervivencia Celular/fisiología , Femenino , Vectores Genéticos , Neuronas/citología , Neuronas/fisiología , Embarazo , Proproteína Convertasas , Ratas , Ratas Sprague-Dawley , Retroviridae/genética , Células Madre/citología , Células Madre/fisiologíaRESUMEN
vax2 is a recently isolated homeobox gene, that plays an important role in controlling the dorso-ventral patterning of the retina. In this paper we present a thorough description of the Xvax2 expression pattern all along Xenopus embryogenesis, and compare this pattern in detail to that shown by Xvax1b and Xpax2, two genes also involved in ventral eye development. At early neurula stages, while Xpax2 starts to be expressed within the eye field, both Xvax2 and Xvax1b are exclusively activated in the presumptive ventral telencephalon. Since midneurula stages, Xvax2 and Xvax1b are also transcribed in the medial aspect of the eye field. At tailbud and tadpole stages, Xvax2, Xvax1b and Xpax2 expression overlaps in the optic stalk and nerve and in the optic disk, while Xvax2 and Xvax1b also display specific activation domains in the ventral retina as well as in the ventral telencephalon and diencephalon. Finally, during metamorphosis a high level of both Xvax2 and Xvax1b transcription is maintained in the optic chiasm. In addition, Xvax1b is transcribed in the ventral hypothalamus and in the hypophysis, whereas a strong Xvax2 expression is retained in the ventral portion of the mature retina.
Asunto(s)
Ojo/embriología , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/genética , Retina/embriología , Telencéfalo/embriología , Proteínas de Xenopus , Xenopus laevis/embriología , Animales , Encéfalo/embriología , ADN Complementario/metabolismo , Embrión no Mamífero/metabolismo , Biblioteca de Genes , Hibridación in Situ , Datos de Secuencia Molecular , Factores de Tiempo , Transcripción GenéticaRESUMEN
Vertebrate eye formation is a complex process which involves early specification of the prospective eye territory, induction events, patterning along the polarity axes and regional specification, to bring about the proper morphogenetic movements, cell proliferation, cell differentiation and neural connections allowing visual function. The molecular machinery underlying such complex developmental events is presently under an intense research scrutiny and many associated genetic factors have been isolated and characterized. These studies produced striking knowledge in the field, especially with respect to uncovering the role of key genes and their possible evolutionary conservation. Presently, a major task is to define the complex interactions connecting the multiplicity of molecular players that regulate eye development. We recently identified two homeobox genes, Xrx1 and Xvax2, and studied their function by using the Xenopus embryo as a developmental model system. Xrx1 and Xvax2 control key aspects of eye development. In particular, Xrx1 appears to play a role in the early specification of anterior neural regions fated to give rise to retina and forebrain structures, and in promoting cell proliferation within these territories. On the other hand, Xvax2 is involved in regulating the eye proximo-distal and/or dorsoventral polarity, and the morphogenetic movements taking place during formation of the optic stalk and cup. Here we review the experimental results addressing the roles of Xrx1 and Xvax2 and their vertebrate orthologues, and discuss their relationship with other molecules also playing a related function in eye development.
Asunto(s)
Ojo/embriología , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox/fisiología , Proteínas de Xenopus , Animales , División Celular , Linaje de la Célula , Proteínas del Ojo , Proteínas de Homeodominio/fisiología , Modelos Biológicos , Prosencéfalo/embriología , Retina/embriología , Xenopus/embriologíaRESUMEN
We describe the cloning, expression pattern and functional overexpression analysis of Xotx5b, a new member of the Otx gene family in Xenopus laevis. Early expression of Xotx5b resembles that of Xotx2, being detected in the organizer region at early gastrula stage, and, shortly after, also in anterior neuroectoderm. During neurula stages Xotx5b exhibits a changing and dynamic pattern of expression. After neural tube closure, Xotx5b is expressed in the eye and pineal gland, both involved in photoreception. Overexpression of Xotx5b has a similar effect to that of Xotx2, producing posterior truncations and inducing ectopic cement gland and neural tissue in whole embryos. In animal cap assays, Xotx5b and Xotx2 are both able to activate XAG, to strongly suppress the expression of the epidermal marker XK81, and to reciprocally activate each other. Finally, in einsteck transplantation assays, Xotx5b is able to respecify a tail/trunk organizer to a head organizer.
Asunto(s)
Ojo/embriología , Proteínas de Homeodominio/genética , Proteínas de Xenopus , Xenopus/embriología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Biomarcadores , Clonación Molecular , Gástrula/metabolismo , Biblioteca de Genes , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/fisiología , Hibridación in Situ , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/biosíntesis , Cresta Neural/metabolismo , Factores de Transcripción Otx , Fenotipo , Plásmidos/metabolismo , ARN Mensajero/metabolismo , ARN Mensajero/farmacología , Factores de Tiempo , Distribución Tisular , Transactivadores/biosíntesis , Xenopus/genéticaRESUMEN
The antiatherogenic effect of estrogen is mediated, in part, by inhibitory effects on endothelial vascular cell adhesion molecule-1 (VCAM-1) expression. To determine the mechanism by which estrogen regulates VCAM-1 expression, we compared the effect of 17beta-estradiol (E(2)) and of the glucocorticoid dexamethasone (Dex) on lipopolysaccharide (LPS)-induced VCAM-1 expression in human endothelial cells. E(2) decreased LPS-induced VCAM-1 mRNA and protein expression to a greater extent than Dex. Dex, but not E(2), stabilized VCAM-1 mRNA. This correlated with inhibition of monocytoid U937 cell adhesion to endothelial cells. Transfection of endothelial cells with a functional VCAM-1 promoter construct showed that E(2) inhibited LPS-induced VCAM-1 gene transcription more potently than did Dex. However, using a truncated construct containing only the nuclear factor-kappaB (NF-kappaB)-responsive elements but lacking the consensus sequences for activator protein-1 (AP-1) and GATA, E(2) and Dex had similar inhibitory effects. Consistently, gel-shift assays showed that E(2) and Dex comparably inhibit LPS-induced activation of NF-kappaB, whereas E(2) inhibited LPS-induced activation of AP-1 and GATA to a greater extent than Dex. E(2) inhibition of NF-kappaB after LPS treatment was associated with decreased inhibitor kappaB (IkappaB) kinase activity and with a stabilization of the NF-kappaB inhibitor IkappaBalpha. These results indicate that E(2) decreases VCAM-1 gene expression through the inhibition of NF-kappaB, AP-1, and GATA and suggest novel mechanisms for the antiatherogenic effect of estrogen on the vascular wall.
Asunto(s)
Dexametasona/farmacología , Endotelio Vascular/efectos de los fármacos , Estradiol/farmacología , Transcripción Genética/efectos de los fármacos , Molécula 1 de Adhesión Celular Vascular/genética , Animales , Bovinos , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Factor de Transcripción GATA2 , Humanos , Proteínas I-kappa B/metabolismo , Lipopolisacáridos/farmacología , Monocitos/efectos de los fármacos , Monocitos/fisiología , FN-kappa B/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/análisis , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/metabolismoRESUMEN
XHNF1(&bgr;) is a homeobox-containing gene initially expressed at the blastula stage in the vegetal part of the Xenopus embryo. We investigated its early role by functional ablation, through mRNA injection of an XHNF1(beta)/engrailed repressor fusion construct (XHNF1(beta)/EngR). Dorsal injections of XHNF1(beta)/EngR mRNA abolish dorsal mesoderm formation, leading to axial deficiencies; ventral injections disrupt ventral mesoderm formation without affecting axial development. XHNF1(beta)/EngR phenotypic effects specifically depend on the DNA-binding activity of its homeodomain and are fully rescued by coinjection of XHNF1(beta) mRNA. Vegetal injection of XHNF1(beta)/EngR mRNA blocks the mesoderm-inducing ability of vegetal explants. Both B-Vg1 and VegT maternal determinants trigger XHNF1(beta) expression in animal caps. XHNF1(beta)/EngR mRNA blocks B-Vg1-mediated, but not by eFGF-mediated, mesoderm induction in animals caps. However, wild-type XHNF1(beta) mRNA does not trigger Xbra expression in animal caps. We conclude that XHNF1(beta) function is essential, though not sufficient, for mesoderm induction in the Xenopus embryo.
Asunto(s)
Proteínas de Unión al ADN/genética , Genes Homeobox , Mesodermo/citología , Factores de Transcripción/genética , Xenopus/embriología , Xenopus/genética , Animales , Secuencia de Bases , Tipificación del Cuerpo/efectos de los fármacos , Tipificación del Cuerpo/genética , Cartilla de ADN/genética , Inducción Embrionaria/efectos de los fármacos , Inducción Embrionaria/genética , Regulación del Desarrollo de la Expresión Génica , Factor Nuclear 1-beta del Hepatocito , Hibridación in Situ , Mesodermo/metabolismo , Microinyecciones , Fenotipo , ARN Mensajero/administración & dosificación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Crecimiento Transformador beta/genética , Xenopus/metabolismo , Proteínas de XenopusRESUMEN
The PC3 gene is transiently expressed during neurogenesis in precursor cells of the telencephalic ventricular/subventricular zone, and is rapidly downregulated before cell migration and differentiation. It is thought to have a role in controlling cell proliferation, but its precise function is not known. Here we present evidence that PC3, when overexpressed in vitro by retroviral-mediated gene transfer, acts by interfering with the normal pattern of cell division. Firstly, we report evidence that PC3 overexpression reduces the rate of cell proliferation in both NIH 3T3 cells and embryonic precursor cells from the rat cerebral cortex. Secondly, when studying the pattern of BrdU dilution in clones of cortical precursors, we observe that clones transduced with PC3 show an asymmetric pattern of BrdU dilution more frequently than clones transduced with a control vector. We discuss the hypothesis that the higher number of PC3 transduced clones showing an asymmetric pattern of BrdU dilution may be due to an increase in asymmetric cell divisions.
Asunto(s)
Corteza Cerebral/citología , Corteza Cerebral/fisiología , Genes Supresores de Tumor , Proteínas Inmediatas-Precoces/biosíntesis , Células 3T3 , Animales , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , Diferenciación Celular , División Celular , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica , Proteínas Inmediatas-Precoces/genética , Ratones , Ratas , Retroviridae , Transfección , Proteínas Supresoras de TumorRESUMEN
We have identified a transcription factor specifically expressed in the developing vertebrate eye. We named this gene vax2 because of the high degree of sequence similarity to the recently described vax1. Both in the human and mouse genomes, vax2 is localized in the vicinity of the emx1 gene. This mapping assignment, together with the previously reported colocalization of Vax1 and Emx2 in mouse, indicates that the vax and the emx genes may be organized in clusters. vax2 has a remarkable expression domain confined to the ventral portion of the prospective neural retina in mouse, human, and Xenopus. The overexpression of either the frog Xvax2 or the human VAX2 in Xenopus embryos leads to an aberrant eye phenotype and, in particular, determines a ventralizing effect on the developing eye. The expression domain of the transcription factor Xpax2, normally confined to the ventral developing retina, extends to the dorsal region of the retina after overexpression of vax2. On the other hand, the expression of Xvent2, a molecular marker of the dorsal retina, is strongly reduced. Furthermore, vax2 overexpression induces a striking expansion of the optic stalk, a structure deriving from the ventralmost region of the eye vesicle. Altogether, these data indicate that vax2 plays a crucial role in eye development and, in particular, in the specification of the ventral optic vesicle.
Asunto(s)
Ojo/embriología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/fisiología , Proteínas de Xenopus , Secuencia de Aminoácidos , Animales , Tipificación del Cuerpo/genética , ADN Complementario/análisis , Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Ligamiento Genético , Humanos , Hibridación in Situ , Larva , Ratones , Datos de Secuencia Molecular , Fenotipo , Mapeo Físico de Cromosoma , Retina/embriología , Retina/metabolismo , Homología de Secuencia de Aminoácido , Factores de Tiempo , Distribución Tisular , XenopusRESUMEN
The anteriormost part of the neural plate is fated to give rise to the retina and anterior brain regions. In Xenopus, this territory is initially included within the expression domain of the bicoid-class homeobox gene Xotx2 but very soon, at the beginning of neurulation, it becomes devoid of Xotx2 transcripts in spatiotemporal concomitance with the transcriptional activation of the paired-like homeobox gene Xrx1. By use of gain- and loss-of-function approaches, we have studied the role played by Xrx1 in the anterior neural plate and its interactions with other anterior homeobox genes. We find that, at early neurula stage Xrx1 is able to repress Xotx2 expression, thus first defining the retina-diencephalon territory in the anterior neural plate. Overexpression studies indicate that Xrx1 possesses a proliferative activity that is coupled with the specification of anterior fate. Expression of a Xrx1 dominant repressor construct (Xrx1-EnR) results in a severe impairment of eye and anterior brain development. Analysis of several brain markers in early Xrx1-EnR-injected embryos reveals that anterior deletions are preceded by a reduction of anterior gene expression domains in the neural plate. Accordingly, expression of anterior markers is abolished or decreased in animal caps coinjected with the neural inducer chordin and the Xrx1-EnR construct. The lack of expansion of mid-hindbrain markers, and the increase of apoptosis in the anterior neural plate after Xrx1-EnR injection, indicate that anterior deletions result from an early loss of anterior neural plate territories rather than posteriorization of the neuroectoderm. Altogether, these data suggest that Xrx1 plays a role in assigning anterior and proliferative properties to the rostralmost part of the neural plate, thus being required for eye and anterior brain development.
Asunto(s)
Encéfalo/embriología , Ojo/embriología , Glicoproteínas , Proteínas de Homeodominio/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Proteínas de Xenopus , Xenopus/embriología , Animales , Apoptosis , Proteínas del Ojo , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox , Hibridación in Situ , Microinyecciones , Proteínas/genética , ARN sin Sentido/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genéticaRESUMEN
We have isolated and studied the expression pattern of Xemx1 and Xemx2 genes in Xenopus laevis. Xemx genes are the homologues of mouse Emx genes, related to Drosophila empty spiracles. They are expressed in selected regions of the developing brain, particularly in the telencephalon, and, outside the brain, in the otic vesicles, olfactory placodes, visceral arches and the developing excretory system. We also report on experiments concerning the tissue and molecular signals responsible for their activation in competent ectoderm. Xemx genes are activated in ectoderm conjugated with head organizer tissue, but not with tail organizer tissue. Furthermore, they are not activated in animal cap either by noggin or by Xnr3, thus suggesting that a different inducer or the integration of several signals may be responsible for their activation.
Asunto(s)
Tipificación del Cuerpo/genética , Inducción Embrionaria/genética , Proteínas de Homeodominio/genética , Telencéfalo/embriología , Xenopus laevis/embriología , Xenopus laevis/genética , Secuencia de Aminoácidos , Animales , Tipificación del Cuerpo/fisiología , Encéfalo/embriología , Encéfalo/metabolismo , Clonación Molecular , Ectodermo/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/biosíntesis , Datos de Secuencia Molecular , Telencéfalo/metabolismo , Factores de TranscripciónRESUMEN
Strand-specific transcripts of a satellite DNA of the newts, Notophthalmus and Triturus, are present in cells in monomeric and multimeric sizes. These transcripts undergo self-catalyzed, site-specific cleavage in vitro: the reaction requires Mg2+ and is mediated by a "hammerhead" domain. Transcription of the newt ribozyme appears to be performed by RNA polymerase II under the control of a proximal sequence element and a distal sequence element. In vitro, the newt ribozyme can cleave in trans an RNA substrate, suggesting that in vivo it might be involved in RNA processing events, perhaps as a riboprotein complex. Here we show that the newt ribozyme is in fact present as a riboprotein particle of about 12 S in the oocytes of Triturus. In addition, reconstitution experiments and gel-shift analyses show that a complex is assembled in vitro on the monomeric ribozyme molecules. UV cross-linking studies identify a few polypeptide species, ranging from 31 to 65 kDa, associated to the newt ribozyme with different affinities. Finally, we find that an appropriate oligoribonucleotide substrate is specifically cleaved by the riboproteic activity in S-100 ovary extracts.
Asunto(s)
ARN Catalítico/química , Ribonucleoproteínas/química , Animales , ADN/genética , ADN/metabolismo , Notophthalmus , ARN Catalítico/genética , ARN Catalítico/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Transcripción Genética , TriturusRESUMEN
To analyse the trans-cleavage activity of the hammerhead ribozyme occurring in the ovary of the newt (Notophthalmus, Triturus) in more detail, six synthetic ribozymes representing natural and modified hammerhead sequences were tested with both short oligoribonucleotides and long transcripts as substrates. The same analysis was also performed with the monomer (330 nucleotides) newt ribozyme and variants thereof. None of the ribozymes comprising the newt natural sequence showed activity under multiple turnover conditions, regardless of sequence changes in stem and loop II. With excess of ribozyme, the same ribozymes cleaved only to a limited extent a short substrate and extremely poorly a target site embedded within a long transcript. The addition of whole ovary cell extract had little influence on cleavage activity of short substrates. However, sequence changes in stems I and III to target different sequences considerably improved cleavage ability of the ribozymes under all conditions used. An RNA secondary-structure folding program showed that ribozymes with the natural newt sequence did not fold in a hammerhead structure whereas those with the changes in stem I and III did. These results suggest that the sequence of the stems I and III impairs the assembly of the newt ribozyme into a bimolecular hammerhead complex in vitro and that proteins present in the ovaries do not facilitate activity.
Asunto(s)
ARN Catalítico/metabolismo , Salamandridae/metabolismo , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Pliegue de Proteína , ARN Catalítico/químicaRESUMEN
We have isolated a novel Xenopus homeobox gene, Xrx1, belonging to the paired-like class of homeobox genes. Xrx1 is expressed in the anterior neural plate, and subsequently in the neural structures of the developing eye (neural retina and pigmented epithelium), and in other forebrain structures deriving from the anterior neural plate: in the pineal gland, throughout its development, in the diencephalon floor and in the hypophysis. Its rostral limit of expression corresponds to the chiasmatic ridge, which some authors consider as the anteriormost limit of the neural tube: thus, Xrx1 may represent one of the most anteriorly expressed homeobox genes reported to date. Moreover, its expression in organs implicated in the establishment of circadian rhythms, may suggest for Xrx1 a role in the genetic control of this function. Finally, analysis of Xrx1 expression in embryos subjected to various treatments, or microinjected with different dorsalizing agents (noggin, Xwnt-8), suggests that vertical inductive signals leading to head morphogenesis are required to activate Xrx1.
Asunto(s)
Ojo/embriología , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox , Proteínas de Homeodominio/genética , Glándula Pineal/embriología , Proteínas de Xenopus , Xenopus laevis/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas del Ojo , Regulación del Desarrollo de la Expresión Génica/genética , Hibridación in Situ , Litio/farmacología , Datos de Secuencia Molecular , Morfogénesis/efectos de los fármacos , Sistema Nervioso/embriología , ARN Mensajero/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Tretinoina/farmacología , Xenopus laevis/embriologíaRESUMEN
The vertebrate Otx gene family is related to otd, a gene contributing to head development in Drosophila. We previously reported on the expression of Xotx2 gene, homologous to the murine Otx2 gene, during early Xenopus development. In the present paper we report an extensive analysis of the expression pattern of Xotx2 during later stages of development and also the cloning and developmental expression of two additional Otx Xenopus genes, Xotx1 and Xotx4. These latter two genes bear a good degree of homology to murine Otx1, higher for Xotx1 than for Xotx4. Both these genes are expressed in the forebrain and midbrain regions and their developmental patterns of expression are very similar, although not perfectly superimposable. Spatial and temporal expression patterns of the three Xotx genes suggest that they may be involved in the early subdivision of the rostral brain, providing antero-posterior positional information within the most anterior districts of the neuraxis. The three Xotx genes are expressed in all the developing sense organs of the head, eyes, olfactory system and otic vesicles. By in situ hybridization the earliest detectable expression is found in anterior mesendoderm for Xotx2, and in presumptive anterior neuroectoderm for Xotx1 and Xotx4. In addition, we examined whether Xotx1 is expressed in exogastrulae, finding that Xotx1 expression can be activated in the apparent absence of vertical signals of neural induction.
Asunto(s)
Encéfalo/embriología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas del Tejido Nervioso/genética , Transactivadores/genética , Xenopus laevis/embriología , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Hibridación in Situ , Datos de Secuencia Molecular , Factores de Transcripción Otx , Proteínas de XenopusRESUMEN
PC4 is an early NGF-inducible gene, transiently expressed during the in vitro differentiation of PC12 cells toward a neuronal phenotype. By in situ hibridization analysis, we found that PC4 is expressed at high levels along the whole neural tube of early rat embryos. PC4 mRNA expression is not uniform across the wall of the neural tube, the autoradiographic signal being most intense on the ventricular layer. At later stages, when the rate of proliferation and production of postmitotic neurons decreases, PC4 gene expression also decreases and becomes restricted to the telencephalon, that is the last region to complete neurogenesis. Thus the expression of PC4 gene, although not exclusive of proliferating cells, appears to be correlated to the time span of proliferation of neuronal and glial precursors.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Genes Inmediatos-Precoces/fisiología , Sistema Nervioso/embriología , Sistema Nervioso/metabolismo , Animales , Autorradiografía , Northern Blotting , División Celular/fisiología , Femenino , Hibridación in Situ , Embarazo , ARN Mensajero/biosíntesis , RatasRESUMEN
We found that deprivation of pattern vision in one eye, that leaves luminance detection performance unaffected, is sufficient to reduce brain-derived neurotrophic factor (but not trkB) messenger RNA in the visual cortex of young and adult rats. Monocular deprivation by means of eyelids' suture was performed during or after the critical period and the cortical amount of brain-derived neurotrophic factor messenger RNA was analysed by in situ hybridization and RNAase protection after 15-30 days of deprivation. A reduction of brain-derived neurotrophic factor messenger RNA was observed in the visual cortex contralateral to the deprived eye in rats monocularly deprived during the critical period. The same reduction was also found in rats monocularly deprived after the end of the critical period, when anatomical or physiological signs of monocular deprivation are absent. The pharmacological blockade of retinal activity equally affected the expression of brain-derived neurotrophic factor messenger RNA in young and adults. Quantitative RNAase protection assays revealed that the cortical level of brain-derived neurotrophic factor messenger RNA was reduced to the same extent when intraocular injections of tetrodotoxin were performed within or after the critical period. A developmental study of brain-derived neurotrophic factor messenger RNA expression in rat visual cortex showed a marked increase around the time of natural eye-opening followed by a plateau from postnatal day 20 until adult age. Messenger RNA for the kinasic domain of brain-derived neurotrophic factor receptor (trkB) was found in the dorsal lateral geniculate nucleus and the visual cortex during development and in adults. Our results suggest that the reduction of brain-derived neurotrophic factor messenger RNA induced by monocular deprivation is related to the absence of pattern vision rather than to the competitive interactions that underlie the effects of monocular deprivation during the critical period.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , ARN Mensajero/metabolismo , Visión Monocular/fisiología , Corteza Visual/metabolismo , Factores de Edad , Animales , Factor Neurotrófico Derivado del Encéfalo , Hibridación in Situ , Proteínas de Microfilamentos/metabolismo , RatasRESUMEN
The expression of the nerve growth factor-inducible gene VGF has been examined by in situ hybridization. Western blot and immunohistochemical studies in the developing and adult rat central nervous system, with particular emphasis on the visual system. Both the messenger RNA and the protein are particularly abundant in the developing dorsal lateral geniculate nucleus, appearing, respectively, at embryonal day 16 and 18. After its onset at E16, VGF messenger RNA expression increases progressively in the dorsal lateral geniculate nucleus and remains high during the first two post-natal weeks; afterwards, it gradually decreases and, at the offset of the plasticity period, it reaches very low levels maintained in adulthood. A similar time course has been observed for VGF protein in the dorsal lateral geniculate nucleus area, by semi-quantitative Western blots. In addition to the presence of the protein in the geniculate neurons, a strong, transient immunoreactivity has been found at the embryonic cortical subplate at E18, reflecting the presence of the antigen in axonal terminals originating from thalamic neurons. Interestingly, we found that the blockade of afferent electrical activity by intraocular injection of tetrodotoxin strongly reduces the level of VGF messenger RNA in the dorsal lateral geniculate nucleus. Although the function of the VGF protein is not known, it had been previously proposed that VGF could be a precursor for neuropeptide/s. The spatiotemporal expression of VGF, together with the observation of a regulation by electrical activity, suggest that this protein may be relevant in the process of synaptogenesis and/or synaptic stabilization in the developing geniculocortical connections.
Asunto(s)
Corteza Cerebral/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Cuerpos Geniculados/metabolismo , Factores de Crecimiento Nervioso/biosíntesis , Factores de Crecimiento Nervioso/genética , Neuronas Aferentes/metabolismo , Sinapsis/fisiología , Vías Aferentes/crecimiento & desarrollo , Vías Aferentes/metabolismo , Animales , Western Blotting , Corteza Cerebral/citología , Corteza Cerebral/crecimiento & desarrollo , Oscuridad , Electrofisiología , Femenino , Cuerpos Geniculados/citología , Cuerpos Geniculados/crecimiento & desarrollo , Inmunohistoquímica , Hibridación in Situ , Embarazo , ARN Mensajero/biosíntesis , Ratas , Tetrodotoxina/toxicidad , Visión Monocular/fisiologíaRESUMEN
In this paper we study Xotx2, a Xenopus homeobox gene related to orthodenticle, a gene expressed in the developing head of Drosophila. The murine cognate, Otx2, is first expressed in the entire epiblast of prestreak embryos and later in very anterior regions of late-gastrulae, including the neuroectoderm of presumptive fore- and mid-brain. In Xenopus, RNase protection experiments reveal that Xotx2 is expressed at low levels throughout early development from unfertilized egg to late blastula, when its expression level significantly increases. Whole-mount in situ hybridization shows a localized expression in the dorsal region of the marginal zone at stage 9.5. At stage 10.25 Xotx2 is expressed in dorsal bottle cells and in cells of the dorsal deep zone fated to give rise to prechordal mesendoderm, suggesting a role in the specification of very anterior structures. In stage 10.5 gastrulae, Xotx2 transcripts start to be detectable also in presumptive anterior neuroectoderm, where they persist in subsequent stages. Various treatments of early embryos cause a general reorganization of Xotx2 expression. In particular, retinoic acid treatment essentially abolishes Xotx2 expression in neuroectoderm. Microinjection of Xotx2 mRNA in 1-, 2- and 4-cell stage embryos causes the appearance of secondary cement glands and partial secondary axes in embryos with reduced trunk and tail structures. The presence of the Xotx2 homeodomain is required to produce these effects. In particular, this homeodomain contains a specific lysine residue at position 9 of the recognition helix. Microinjected transcripts of Xotx2 constructs containing a homeodomain where this lysine is substituted by a glutamine or a glutamic acid residue fail to cause these effects.
Asunto(s)
Genes Homeobox , Proteínas de Homeodominio , Mesodermo/fisiología , Proteínas del Tejido Nervioso/genética , Transactivadores/genética , Xenopus laevis/genética , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Blastocisto/fisiología , Femenino , Gástrula/fisiología , Expresión Génica/efectos de los fármacos , Expresión Génica/efectos de la radiación , Ratones , Datos de Secuencia Molecular , Morfogénesis/efectos de los fármacos , Morfogénesis/efectos de la radiación , Factores de Transcripción Otx , Óvulo/fisiología , Fenotipo , Homología de Secuencia de Aminoácido , Tretinoina/farmacología , Rayos Ultravioleta , Proteínas de Xenopus , Xenopus laevis/embriología , Cigoto/fisiologíaRESUMEN
We examined the developmental expression of PC3, a nerve growth factor (NGF) early induced gene in PC12 cells, in the rat central nervous system (CNS) and we found that it represents a molecular marker of ongoing postmitotic neurons production. PC3 is initially expressed in the ventral quarter of the neural tube, at the level of the presumptive cervical spinal cord just where and when (10-11 days post coitum (dpc)) the motor neurons are arising. Subsequently, the appearance of PC3 expression follows a ventro-dorsal and a rostro-caudal gradient in the spinal cord and a caudo-rostral gradient across the brain vesicles that coincide, both spatially and temporally, with the gradients of neurogenesis described in the literature. As in PC12 cells, PC3 mRNA expression appears to be transient in vivo. In all regions of the CNS, it is restricted to the ventricular zone of the neuroepithelium, while neuronal precursors cease to express PC3 as they migrate to the mantle zone. Moreover, PC3 mRNA disappears from the various regions of the CNS as neurogenesis ceases.