RESUMEN
PURPOSE: A time-course analysis of gene regulation in the adult rat retina after intraorbital nerve crush (IONC) and intraorbital nerve transection (IONT). METHODS: RNA was extracted from adult rat retinas undergoing either IONT or IONC at increasing times post-lesion. Affymetrix RAE230.2 arrays were hybridized and analyzed. Statistically regulated genes were annotated and functionally clustered. Arrays were validated by means of quantative reverse transcription polymerase chain reaction (qRT-PCR) on ten regulated genes at two times post-lesion. Western blotting and immunohistofluorescence for four pro-apoptotic proteins were performed on naïve and injured retinas. Finally, custom signaling maps for IONT- and IONC-induced death response were generated (MetaCore, Genego Inc.). RESULTS: Here we show that over time, 3,219 sequences were regulated after IONT and 1,996 after IONC. Out of the total of regulated sequences, 1,078 were commonly regulated by both injuries. Interestingly, while IONT mainly triggers a gene upregulation-sustained over time, IONC causes a transitory downregulation. Functional clustering identified the regulation of high interest biologic processes, most importantly cell death wherein apoptosis was the most significant cluster. Ten death-related genes upregulated by both injuries were used for array validation by means of qRT-PCR. In addition, western blotting and immunohistofluorescence of total and active Caspase 3 (Casp3), tumor necrosis factor receptor type 1 associated death domain (TRADD), tumor necrosis factor receptor superfamily member 1a (TNFR1a), and c-fos were performed to confirm their protein regulation and expression pattern in naïve and injured retinas. These analyses demonstrated that for these genes, protein regulation followed transcriptional regulation and that these pro-apoptotic proteins were expressed by retinal ganglion cells (RGCs). MetaCore-based death-signaling maps show that several apoptotic cascades were regulated in the retina following optic nerve injury and highlight the similarities and differences between IONT and IONC in cell death profiling. CONCLUSIONS: This comprehensive time course retinal transcriptome study comparing IONT and IONC lesions provides a unique valuable tool to understand the molecular mechanisms underlying optic nerve injury and to design neuroprotective protocols.
Asunto(s)
Perfilación de la Expresión Génica , Compresión Nerviosa , Traumatismos del Nervio Óptico/genética , Nervio Óptico/metabolismo , Nervio Óptico/patología , Retina/metabolismo , Retina/patología , Animales , Muerte Celular , Análisis por Conglomerados , Femenino , Regulación de la Expresión Génica , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
The Drosophila wings-up A gene encodes Troponin I. Two regions, located upstream of the transcription initiation site (upstream regulatory element) and in the first intron (intron regulatory element), regulate gene expression in specific developmental and muscle type domains. Based on LacZ reporter expression in transgenic lines, upstream regulatory element and intron regulatory element yield identical expression patterns. Both elements are required for full expression levels in vivo as indicated by quantitative reverse transcription-polymerase chain reaction assays. Three myocyte enhancer factor-2 binding sites have been functionally characterized in each regulatory element. Using exon specific probes, we show that transvection is based on transcriptional changes in the homologous chromosome and that Zeste and Suppressor of Zeste 3 gene products act as repressors for wings-up A. Critical regions for transvection and for Zeste effects are defined near the transcription initiation site. After in silico analysis in insects (Anopheles and Drosophila pseudoobscura) and vertebrates (Ratus and Coturnix), the regulatory organization of Drosophila seems to be conserved. Troponin I (TnI) is expressed before muscle progenitors begin to fuse, and sarcomere morphogenesis is affected by TnI depletion as Z discs fail to form, revealing a novel developmental role for the protein or its transcripts. Also, abnormal stoichiometry among TnI isoforms, rather than their absolute levels, seems to cause the functional muscle defects.