RESUMEN
In Assisted Reproductive Technology (ART), the pregnancy and birth rates following in vitro fertilization (IVF) attempts are still low. Recently, research in the field of ART has explored new oocyte and embryo quality selection criteria such as apoptotic markers. Many studies provided evidence that bad oocyte and embryo quality can be associated with apoptosis. The aim of this review is to summarize our current knowledge on the apoptosis process in oocytes and embryos, and focus on the possibility of using apoptotic markers as a reliable and predictive marker to select competent oocytes and embryos during IFV.
Asunto(s)
Apoptosis , Blastocisto/citología , Blastocisto/patología , Oocitos/citología , Oocitos/patología , Biomarcadores , Implantación del Embrión/fisiología , Desarrollo Embrionario , Femenino , Enfermedades Fetales/genética , Enfermedades Fetales/patología , Humanos , Oocitos/fisiología , Ovulación/fisiología , Embarazo , Diagnóstico Prenatal , Técnicas Reproductivas Asistidas/tendenciasRESUMEN
We have adapted the whole-mount in situ hybridization technique to perform high-throughput gene expression analysis in mouse embryos. A large-scale screen for genes showing specific expression patterns in the mid-gestation embryo was carried out, and a large number of genes controlling development were isolated. From 35760 clones of a 9.5 d.p.c. cDNA library, a total of 5348 cDNAs, enriched for rare transcripts, were selected and analyzed by whole-mount in situ hybridization. Four hundred and twenty-eight clones revealed specific expression patterns in the 9.5 d.p.c. embryo. Of 361 tag-sequenced clones, 198 (55%) represent 154 known mouse genes. Thirty-nine (25%) of the known genes are involved in transcriptional regulation and 33 (21%) in inter- or intracellular signaling. A large number of these genes have been shown to play an important role in embryogenesis. Furthermore, 24 (16%) of the known genes are implicated in human disorders and three others altered in classical mouse mutations. Similar proportions of regulators of embryonic development and candidates for human disorders or mouse mutations are expected among the 163 new mouse genes isolated. Thus, high-throughput gene expression analysis is suitable for isolating regulators of embryonic development on a large-scale, and in the long term, for determining the molecular anatomy of the mouse embryo. This knowledge will provide a basis for the systematic investigation of pattern formation, tissue differentiation and organogenesis in mammals.
Asunto(s)
Desarrollo Embrionario y Fetal/genética , Pruebas Genéticas/métodos , Animales , Secuencia de Bases , Cartilla de ADN/genética , ADN Complementario/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Hibridación in Situ , Ratones , MutaciónRESUMEN
Floor plate cells at the midline of the neural tube are specified by high-level activity of Sonic hedgehog (Shh) secreted by notochord, whereas motor neurons are thought to be specified by a lower level activity of Shh secreted in turn by floor plate cells. In Drosophila, the Gli zinc finger protein Cubitus interruptus functions as a transcription factor activating Hedgehog-responsive genes. We report that the expression of known Shh-responsive genes such as Ptc and Gli1 is downregulated in mutant mice lacking Gli2 function. Gli2 mutants fail to develop a floor plate yet still develop motor neurons, which occupy the ventral midline of the neural tube. Our results imply that Gli2 is required to mediate high level but not low level Shh activity and show that the development of motor neurons can occur in the absence of floor plate induction.
Asunto(s)
Diferenciación Celular/genética , Proteínas de Homeodominio , Proteínas del Tejido Nervioso , Proteínas/metabolismo , Proteínas/fisiología , Proteínas Represoras , Transducción de Señal/fisiología , Transactivadores , Proteínas de Xenopus , Animales , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo/fisiología , Embrión de Mamíferos/citología , Proteínas del Ojo , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Hedgehog , Factor Nuclear 3-beta del Hepatocito , Factores de Transcripción de Tipo Kruppel , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Neuronas Motoras/fisiología , Mutación/genética , Cresta Neural/crecimiento & desarrollo , Notocorda/crecimiento & desarrollo , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas/análisis , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box , Receptores Patched , Receptor Patched-1 , Receptores de Superficie Celular , Factores de Transcripción/análisis , Proteína con Dedos de Zinc GLI1 , Proteína Gli2 con Dedos de Zinc , Proteína Gli3 con Dedos de ZincRESUMEN
Shp-1, Shp-2 and corkscrew comprise a small family of cytoplasmic tyrosine phosphatases that possess two tandem SH2 domains. To investigate the biological functions of Shp-2, a targeted mutation has been introduced into the murine Shp-2 gene, which results in an internal deletion of residues 46-110 in the N-terminal SH2 domain. Shp-2 is required for embryonic development, as mice homozygous for the mutant allele die in utero at mid-gestation. The Shp-2 mutant embryos fail to gastrulate properly as evidenced by defects in the node, notochord and posterior elongation. Biochemical analysis of mutant cells indicates that Shp-2 can function as either a positive or negative regulator of MAP kinase activation, depending on the specific receptor pathway stimulated. In particular, Shp-2 is required for full and sustained activation of the MAP kinase pathway following stimulation with fibroblast growth factor (FGF), raising the possibility that the phenotype of Shp-2 mutant embryos results from a defect in FGF-receptor signalling. Thus, Shp-2 modulates tyrosine kinase signalling in vivo and is crucial for gastrulation during mammalian development.
Asunto(s)
Tipificación del Cuerpo/genética , Mesodermo/enzimología , Proteínas Tirosina Fosfatasas/genética , Dominios Homologos src , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Endotelio Vascular/embriología , Endotelio Vascular/enzimología , Activación Enzimática/genética , Factores de Crecimiento de Fibroblastos/farmacología , Gástrula/enzimología , Genes Letales , Péptidos y Proteínas de Señalización Intracelular , Ratones , Mutación , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Tirosina Fosfatasas con Dominio SH2 , Transducción de Señal/genéticaRESUMEN
We have used a gene-trap vector and mouse embryonic stem (ES) cells to screen for insertional mutations in genes developmentally regulated at 8.5 days of embryogenesis (dpc). From 38,730 cell lines with vector insertions, 393 clonal integrations had disrupted active transcription units, as assayed by beta-galactosidase reporter gene expression. From these lines, 290 clones were recovered and injected into blastocysts to assay for reporter gene expression in 8.5-dpc chimeric mouse embryos. Of these, 279 clones provided a sufficient number of chimeric embryos for analysis. Thirty-six (13%) showed restricted patterns of reporter-gene expression, 88 (32%) showed widespread expression and 155 (55%) failed to show detectable levels of expression. Further analysis showed that approximately one-third of the clones that did not express detectable levels of the reporter gene at 8.5 dpc displayed reporter gene activity at 12.5 dpc. Thus, a large proportion of the genes that are expressed in ES cells are either temporally or spatially regulated during embryogenesis. These results indicate that gene-trap mutageneses in embryonic stem cells provide an effective approach for isolating mutations in a large number of developmentally regulated genes.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Pruebas Genéticas/métodos , Mutación , Animales , Blastocisto , Quimera , Genes Reporteros/genética , Vectores Genéticos/genética , Ratones , Mutagénesis Insercional , Células Madre , beta-Galactosidasa/genéticaRESUMEN
We have used a gene trap (GT) vector and embryonic stem (ES) cell chimeras to screen for insertions of the lacZ reporter gene into transcription units that are spatially and temporally regulated during early mouse embryogenesis. GT vectors which can act as both a reporter and a mutagen have been previously used to isolate new genes that are essential for mouse development. In this paper we describe a GT insertion which displays a very restricted pattern of expression in the gastrulating embryo. beta-Galactosidase activity was first detected at 7.5 days post-coitum (E7.5) in the node region of the embryo and extended to the midline structures at E8.0. At E9.5 expression was restricted to the floor plate, the notochord, the roof of the gut, and the liver anlage. Expression appeared in the somites at E10.0 and later became more widespread. We used rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) to clone a partial 360 base pair (bp) cDNA representing an endogenous sequence and containing an open reading frame (ORF) fused in frame to the lacZ reporter gene. The sequence showed no homology to any known protein or protein domain. An overlapping 1,200 bp fragment from a wild-type cDNA library was cloned and it detected the same pattern of expression as the reporter gene in E7.5, E8.5, and E9.5 wild-type embryos. It hybridized to a 5.4 kb lacZ fusion transcript and to an endogenous transcript of 6.5 kb. The gene was mapped to chromosome 11 and was named cordon-bleu (cobl). No phenotype was detected in mice homozygous for the insertion. However, the insertion may not cause a complete disruption of the gene function. The pattern of expression of cobl is very similar to that of hepatic nuclear factor 3 beta (HNF3 beta) and sonic hedgehog (Shh), both of which are involved in axial patterning. Therefore, the product of the cobl gene may also prove to be an important component of the genetic pathway regulating vertebrate axis formation.