RESUMEN
The use of genetics in recent years has brought to light the need to reevaluate the classification of many gorgonian octocorals. This study focuses on two Leptogorgia species-Leptogorgia virgulata and Leptogorgia hebes-from the northwestern Gulf of Mexico (GOM). We target complete mitochondrial genomes and mtMutS sequences, and integrate this data with previous genetic research of gorgonian corals to resolve phylogenetic relationships and estimate divergence times. This study contributes the first complete mitochondrial genomes for L. ptogorgia virgulata and L. hebes. Our resulting phylogenies stress the need to redefine the taxonomy of the genus Leptogorgia in its entirety. The fossil-calibrated divergence times for Eastern Pacific and Western Atlantic Leptogorgia species based on complete mitochondrial genomes shows that the use of multiple genes results in estimates of more recent speciation events than previous research based on single genes. These more recent divergence times are in agreement with geologic data pertaining to the formation of the Isthmus of Panama.
RESUMEN
PURPOSE: Circadian rhythms are central to vision and retinal physiology. A circadian clock located within the retina controls various rhythmic processes including melatonin synthesis in photoreceptors. In the present study, we evaluated the rhythmic expression of clock genes and clock output genes in retinal explants maintained for several days in darkness. METHODS: Retinas were dissected from Wistar rats, either wild-type or from the Per1-luciferase transgenic line housed under a daily 12 h:12 h light-dark cycle (LD12/12), and put in culture at zeitgeber time (ZT) 12 on semipermeable membranes. Explants from wild-type rats were collected every 4 h over 3 days, and total RNA was extracted, quantified, and reverse transcribed. Gene expression was assessed with quantitative PCR, and the periodicity of the relative mRNA amounts was assessed with nonlinear least squares fitting to sine wave functions. Bioluminescence in explants from Per1-luciferase rats was monitored for several days under three different culture protocols. RESULTS: Rhythmic expression was found for all studied clock genes and for clock downstream targets such as c-fos and arylalkylamine N-acetyltransferase (Aanat) genes. Clock and output genes cycled with relatively similar periods and acrophases (peaks of expression during subjective night, except c-fos, which peaked around the end of the subjective day). Data for Per1 were confirmed with bioluminescence monitoring, which also permitted culture conditions to be optimized to study the retina clock. CONCLUSIONS: Our work shows the free-running expression profile of multiple clock genes and potential clock targets in mammalian retinal explants. This research further strengthens the notion that the retina contains a self-sustained oscillator that can be functionally characterized in organotypic culture.
Asunto(s)
Proteínas CLOCK/genética , Ritmo Circadiano/genética , Regulación de la Expresión Génica , Retina/metabolismo , Técnicas de Cultivo de Tejidos , Animales , Relojes Biológicos/genética , Proteínas CLOCK/metabolismo , Muerte Celular/efectos de los fármacos , Ritmo Circadiano/efectos de los fármacos , Medios de Cultivo/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Luciferasas/metabolismo , Mediciones Luminiscentes , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Retina/citología , Retina/efectos de los fármacos , Factores de TiempoRESUMEN
Axonal regeneration is an essential condition to re-establish functional neuronal connections in the injured adult central nervous system (CNS), but efficient regrowth of severed axons has proven to be very difficult to achieve. Although significant progress has been made in identifying the intrinsic and extrinsic mechanisms involved, many aspects remain unresolved. Axonal development in embryonic CNS (hippocampus) requires the obligate activation of the insulin-like growth factor 1 receptor (IGF-1R). Based on known similarities between axonal growth in fetal compared to mature CNS, we decided to examine the expression of the IGF-1R, using an antibody to the ßgc subunit or a polyclonal anti-peptide antibody directed to the IGF-R (C20), in an in vitro model of adult CNS axonal regeneration, namely retinal ganglion cells (RGC) derived from adult rat retinas. Expression of both ßgc and the ß subunit recognized by C20 antibody were low in freshly isolated adult RGC, but increased significantly after 4 days in vitro. As in embryonic axons, ßgc was localised to distal regions and leading growth cones in RGC. IGF-1R-ßgc co-localised with activated p85 involved in the phosphatidylinositol-3 kinase (PI3K) signaling pathway, upon stimulation with IGF-1. Blocking experiments using either an antibody which neutralises IGF-1R activation, shRNA designed against the IGF-1R sequence, or the PI3K pathway inhibitor LY294002, all significantly reduced axon regeneration from adult RGC in vitro (â¼40% RGC possessed axons in controls vs 2-8% in the different blocking studies). Finally, co-transfection of RGC with shRNA to silence IGF-1R together with a vector containing a constitutively active form of downstream PI3K (p110), fully restored axonal outgrowth in vitro. Hence these data demonstrate that axonal regeneration in adult CNS neurons requires re-expression and activation of IGF-1R, and targeting this system may offer new therapeutic approaches to enhancing axonal regeneration following trauma.
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Axones/fisiología , Sistema Nervioso Central/crecimiento & desarrollo , Receptor IGF Tipo 1/metabolismo , Regeneración , Animales , Axones/efectos de los fármacos , Axones/metabolismo , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/metabolismo , Cromonas/farmacología , Regulación de la Expresión Génica , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Morfolinas/farmacología , Neuronas/citología , Neuronas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Ratas , Ratas Wistar , Receptor IGF Tipo 1/genética , Regeneración/efectos de los fármacos , Regeneración/fisiología , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/fisiología , Transducción de Señal/efectos de los fármacos , Activación TranscripcionalRESUMEN
PURPOSE: Retinal ganglion cells (RGCs) expressing the photopigment melanopsin (Opn4) display intrinsic photosensitivity. In this study, the presence of nonvisual phototransduction cascade components in the developing chicken retina and primary RGCs cultures was investigated, focusing on the two Opn4 genes: the Xenopus (Opn4x) and the mammalian (Opn4m) orthologs. METHODS: Retinas were dissected at different embryonic (E) and postnatal (P) days, and primary RGC cultures were obtained at E8 and kept for 1 hour to 5 days. Samples were processed for RT-PCR and immunochemistry. RESULTS: Embryonic retinas expressed the master eye gene Pax6, the prospective RGC specification gene Brn3, and components of the nonvisual phototransduction cascade, such as Opn4m and the G protein q (Gq) mRNAs at very early stages (E4-E5). By contrast, expression of photoreceptor cell markers (CRX, red-opsin, rhodopsin, and α-transducin) was observed from E7 to E12. Opn4m protein was visualized in the whole retina as early as E4 and remained elevated from E6 to the postnatal days, whereas Opn4x was weakly detected at E8 and highly expressed after E11. RGC cultures expressed Gq mRNA, as well as both Opn4 mRNAs and proteins. Opn4m was restricted exclusively to the GC layer at all ages, whereas Opn4x was limited to the forming GC layer and optic nerve at E8, but by E15, its expression was mostly in Prox1(+) horizontal cells. CONCLUSIONS: The early expression onset of nonvisual phototransduction molecules could confer premature photosensitivity to RGCs, while the appearance of Opn4x expression in horizontal cells suggests the identification of a novel type of photosensitive cell in birds.