RESUMEN
The reticulons are a recently discovered family of proteins that have a predominant localisation to the membrane of the endoplasmic reticulum. The precise function of the reticulons is unclear despite their presence in a wide variety of eukaryotic organisms. Here we describe the characterisation of the Drosophila reticulon, reticulon-like1 (Rtnl1), which is the only functional reticulon in Drosophila. The Rtnl1 locus produces seven predicted mRNA transcripts encoding five different protein isoforms. The different transcripts have tissue-specific expression patterns remarkably similar to their mammalian counterparts. Rtnl1 protein is associated with organelles of the secretory pathway including the endoplasmic reticulum and the Golgi apparatus. Rtnl1 function appears to be non-essential or redundant since loss of function Rtnl1 mutants are viable. However, a significant reduction in life expectancy was seen in Rtnl1 mutant flies. This may point towards a possible protective role for reticulons against conditions of environmental stress.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila/embriología , Retículo Endoplásmico/metabolismo , ARN Mensajero/metabolismo , Animales , Transporte Biológico , Drosophila/genética , Proteínas de Drosophila/metabolismo , Datos de Secuencia Molecular , Mutación , Señales de Clasificación de Proteína , Distribución TisularRESUMEN
Contrary to our knowledge of the genetic control of midline crossing, the mechanisms that generate and maintain the longitudinal axon pathways of the Drosophila CNS are largely unknown. The longitudinal pathways are formed by ipsilateral pioneer axons and the longitudinal glia. The longitudinal glia dictate these axonal trajectories and provide trophic support to later projecting follower neurons. Follower interneuron axons cross the midline once and join these pathways to form the longitudinal connectives. Once on the contralateral side, longitudinal axons are repelled from recrossing the midline by the midline repulsive signal Slit and its axonal receptor Roundabout. We show that longitudinal glia also transiently express roundabout, which halts their ventral migration short of the midline. Once in contact with axons, glia cease to express roundabout and become dependent on neurons for their survival. Trophic support and cell-cell contact restrict glial movement and axonal trajectories. The significance of this relationship is revealed when neuron-glia interactions are disrupted by neuronal ablation or mutation in the glial cells missing gene, which eliminates glia, when axons and glia cross the midline despite continued midline repellent signalling.
Asunto(s)
Sistema Nervioso Central/embriología , Drosophila melanogaster/embriología , Animales , Axones/ultraestructura , Adhesión Celular , Movimiento Celular , Sistema Nervioso Central/citología , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Genes de Insecto , Modelos Biológicos , Mutación , Proteínas del Tejido Nervioso , Neuroglía/citología , Fenotipo , Receptores Inmunológicos/genética , Transducción de Señal , Proteínas RoundaboutRESUMEN
A key feature of the central nervous system of most higher organisms is their bilateral symmetry about the midline. The specialised cells that lie at the midline have an essential role in regulating the axon guidance decisions of both neurons that project axons across the midline and those that project on one side. The midline cells produce both attractive and repellent short- and long-range signals to guide axonal growth. The axons themselves express specific receptors that can be dynamically regulated in response to midline-derived signals. In this way, axons extend toward or away from the midline and those that do cross change their behaviour to respond to longitudinal signals on the contralateral side.
Asunto(s)
Axones/ultraestructura , Sistema Nervioso Central/embriología , Animales , Axones/fisiología , Moléculas de Adhesión Celular Neuronal/fisiología , Sistema Nervioso Central/fisiología , Sistema Nervioso Central/ultraestructura , Drosophila/embriología , Drosophila/fisiología , Drosophila/ultraestructura , Ratones , Factores de Crecimiento Nervioso/fisiología , Transducción de Señal , Pez Cebra/anatomía & histología , Pez Cebra/embriología , Pez Cebra/fisiologíaRESUMEN
Remarkable advances have been made recently to identify the molecules required for the development of neural connections within the brain. A range of ligands and receptors have been uncovered that guide extending neurons to appropriate targets and away from inappropriate areas. These molecules point to the signalling mechanisms that guide the neurons and provide entry points for the further dissection of this process. Here I highlight the part genetic screens and analyses have played in revealing some of the key players in neuronal guidance.
Asunto(s)
Axones , Neuronas/citología , Regulación del Desarrollo de la Expresión GénicaRESUMEN
Asymmetric cell division is a widespread mechanism in developing tissues that leads to the generation of cell diversity. In the embryonic central nervous system of Drosophila melanogaster, secondary precursor cells-ganglion mother cells (GMCs)-divide and produce postmitotic neurons that take on different cell fates. In this study, we show that binary fate decision of two pairs of sibling neurons is accomplished through the interplay of Notch (N) signaling and the intrinsic fate determinant Numb. We show that GMCs have apical-basal polarity and Numb localization and the orientation of division are coordinated to segregate Numb to only one sibling cell. The correct positioning of Numb and the proper orientation of division require Inscuteable (Insc). Loss of insc results in the generation of equivalent sibling cells. Our results provide evidence that sibling neuron fate decision is nonstochastic and normally depends on the presence of Numb in one of the two siblings. Moreover, our data suggest that the fate of some sibling neurons may be regulated by signals that do not require lateral interaction between the sibling cells.
Asunto(s)
Sistema Nervioso Central/embriología , Proteínas del Citoesqueleto/metabolismo , Proteínas de Drosophila , Drosophila melanogaster/embriología , Ganglios de Invertebrados/fisiología , Neuronas/citología , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Diferenciación Celular/fisiología , División Celular/fisiología , Linaje de la Célula/genética , Núcleo Celular/genética , Núcleo Celular/fisiología , Tamaño de la Célula/genética , Tamaño de la Célula/fisiología , Ganglios de Invertebrados/citología , Ganglios de Invertebrados/embriología , Proteínas de Insectos/genética , Proteínas de Insectos/fisiología , Hormonas Juveniles/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Proteínas de Microfilamentos , Mutación/genética , Neuropéptidos , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Receptores Notch , Transducción de Señal/fisiología , Procesos EstocásticosRESUMEN
commissureless and roundabout lead to complementary mutant phenotypes in which either too few or too many axons cross the midline. The robo;comm double-mutant phenotype is identical to robo alone, suggesting that in the absence of robo, comm is no longer required. Comm is expressed on midline cells; Robo is expressed in a dynamic fashion on growth cones and appears to function as an axon guidance receptor. robo function is dosage-sensitive. Overexpression of comm is also dosage-sensitive and leads to a phenotype identical to robo loss-of-function. Comm controls Robo expression; increasing Comm leads to a reduction of Robo protein. The levels of Comm and Robo appear to be tightly regulated to assure that only certain growth cones cross the midline and that those growth cones that do cross never do so again.
Asunto(s)
Axones/fisiología , Sistema Nervioso Central/fisiología , Proteínas de Drosophila , Drosophila/genética , Drosophila/fisiología , Dosificación de Gen , Mutación/fisiología , Receptores Inmunológicos/genética , Receptores Inmunológicos/fisiología , Animales , Expresión Génica/fisiología , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso , Fenotipo , Proteínas RoundaboutRESUMEN
The robo gene in Drosophila was identified in a large-scale mutant screen for genes that control the decision by axons to cross the CNS midline. In robo mutants, too many axons cross and recross the midline. Here we show that robo encodes an axon guidance receptor that defines a novel subfamily of immunoglobulin superfamily proteins that is highly conserved from fruit flies to mammals. For those axons that never cross the midline, Robo is expressed on their growth cones from the outset; for the majority of axons that do cross the midline, Robo is expressed at high levels on their growth cones only after they cross the midline. Transgenic rescue experiments reveal that Robo can function in a cell-autonomous fashion. Robo appears to function as the gatekeeper controlling midline crossing.
Asunto(s)
Axones/fisiología , Secuencia Conservada/genética , Drosophila/embriología , Receptores Inmunológicos/genética , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Sistema Nervioso Central/embriología , Paseo de Cromosoma , Clonación Molecular , Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Genes de Insecto/genética , Inmunoglobulinas/genética , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso , Polimorfismo de Longitud del Fragmento de Restricción , ARN Mensajero/análisis , Ratas , Receptores Inmunológicos/análisis , Receptores Inmunológicos/química , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Proteínas RoundaboutRESUMEN
Diffusible and substrate-bound cues can guide axonal pathway choice via attractive and repulsive signals. A number of families of signalling molecules have been identified, including netrins and their receptors, semaphorins, neuropilins, Eph receptor tyrosine kinases, ephrins and CAMs. Many of these signalling molecules can have a dual role, functioning either as attractants or as repellents. Direction of growth cone extension requires reorganization of the cytoskeleton, which may be directed by the Rho family of GTPases.
Asunto(s)
Sistema Nervioso/crecimiento & desarrollo , Animales , Axones/fisiología , Humanos , Factores de Crecimiento Nervioso/fisiología , Proteínas del Tejido Nervioso/fisiología , Fenómenos Fisiológicos del Sistema Nervioso , Receptores de Netrina , Moléculas de Adhesión de Célula Nerviosa/fisiología , Vías Nerviosas/crecimiento & desarrollo , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptor EphA1 , Receptores de Superficie Celular/fisiología , Transducción de SeñalRESUMEN
The commissureless (comm) gene was identified previously in a large-scale screen for mutations that disrupt CNS axon pathways in Drosophila. The comm gene has a unique mutant phenotype: the complete absence of most axon commissures, while midline cells and other aspects of CNS fate and patterning are left unchanged. Here, we report on the molecular cloning, characterization, and expression of the comm gene. comm encodes a novel protein of 370 amino acids that lacks a signal sequence, has a transmembrane domain, and biochemically copurifies with membranes. COMM mRNA and COMM protein are dynamically expressed during embryogenesis, including by CNS midline glia during the formation of the axon commissures. Anti-COMM antibodies reveal strong staining of organelles likely to include the Golgi complex and endosomes and weaker staining of the cell surface. As commissural growth cones contact and traverse the CNS midline, COMM protein is apparently transferred from midline glia to commissural axons.
Asunto(s)
Axones/fisiología , Sistema Nervioso Central/fisiología , Proteínas de la Membrana/genética , Animales , Secuencia de Bases , Drosophila , Expresión Génica/genética , Inmunohistoquímica , Datos de Secuencia MolecularRESUMEN
The pharmacokinetic properties and relative bioequivalence of two formulations of the antiepileptic Epilim were compared in a three-period, repeat dose, randomized crossover study in 18 male volunteers. A daily dose of 1000 mg of antiepileptic was given either as twice-daily enteric-coated sodium valproate tablets or as twice- or once-daily controlled release tablets (Epilim Chrono, sodium valproate/valproic acid mixture). All regimens were bioequivalent with respect to area under the curve and elimination half-life. The twice-daily controlled release formulation showed reduced mean peak plasma valproate levels and raised mean trough levels compared with the enteric coated tablets. The once-daily controlled release regimen gave reduced mean peak levels but similar mean trough levels to the twice-daily enteric-coated regimen. No major differences between regimens were observed in the time at which peak concentration was observed. Both formulations were well tolerated. The most frequently reported adverse event was mild diarrhoea (all on Chrono treatment) which is probably related to the lack of enteric coating on this formulation. The reduced mean peak-trough variation in plasma valproate levels observed with the twice-daily controlled release regimen is likely to reduce further the low incidence of concentration-related side-effects with sodium valproate and permit more reliable plasma level monitoring. This study also indicates that once-daily treatment with Epilim controlled release would be a suitable replacement for twice-daily dosing with either formulation. Once-daily treatment is likely to give considerable benefits both in convenience and in better patient compliance.
Asunto(s)
Anticonvulsivantes/farmacocinética , Ácido Valproico/farmacocinética , Adolescente , Adulto , Anticonvulsivantes/administración & dosificación , Estudios Cruzados , Preparaciones de Acción Retardada , Esquema de Medicación , Combinación de Medicamentos , Semivida , Humanos , Comprimidos Recubiertos , Ácido Valproico/administración & dosificaciónRESUMEN
The glial cells missing (gcm) gene in Drosophila encodes a novel nuclear protein that is transiently expressed early in the development of nearly all glia. In loss-of-function gcm mutant alleles, nearly all glia fail to differentiate, and, where we can follow them in the PNS, are transformed into neurons. In gain-if-function gcm conditions using transgenic constructs that drive ectopic gcm expression, many presumptive neurons are transformed into glia. Thus, gcm appears to function as a binary genetic switch for glia versus neurons. In the presence of gcm protein, presumptive neurons become glia, while in its absence, presumptive glia become neurons.
Asunto(s)
Drosophila/genética , Genes de Insecto/fisiología , Neuroglía/citología , Neuronas/citología , Secuencia de Aminoácidos , Animales , Diferenciación Celular/genética , Sistema Nervioso Central/embriología , Sistema Nervioso Central/fisiología , Clonación Molecular , Regulación del Desarrollo de la Expresión Génica/fisiología , Datos de Secuencia Molecular , Mutación/fisiología , Neuroglía/fisiología , Neuronas/fisiología , Proteínas Nucleares/genética , Sistema Nervioso Periférico/embriología , Sistema Nervioso Periférico/fisiologíaRESUMEN
We describe an unusual Antennapedia class homeobox gene from the grasshopper Schistocerca gregaria (Orthoptera, African Plague Locust). Its sequence is not sufficiently similar to that of any other insect Hom-Hox gene to identify it unambiguously, but short conserved elements suggest a relationship to the segmentation gene fushi-tarazu, (ftz). We term it Sg Dax (divergent Antennapedia class homeobox gene). Antibodies raised against the protein encoded by this gene reveal two phases of expression during embryogenesis. In the early embryo, it is a marker for the posterior part of the forming embryonic primordium, and subsequently for the posterior part of the growing germ band. In older embryos, it labels a subset of neural precursor cells in each trunk segment, very similar to that defined by the expression of fushi tarazu (ftz) in Drosophila. We suggest that Schistocerca Dax and Drosophila ftz are homologous members of a gene family whose members are diverging relatively rapidly, both in terms of sequence and role in early development.
Asunto(s)
Genes Homeobox/genética , Genes de Insecto/genética , Saltamontes/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sistema Nervioso Central/embriología , Cartilla de ADN/genética , Drosophila/genética , Expresión Génica/fisiología , Saltamontes/embriología , Hibridación in Situ , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
We performed a large-scale screen for mutations that affect the development of CNS axon pathways in the Drosophila embryo. We screened embryos from over 13,500 balanced lines and saved over 250 mutant lines whose phenotypes suggest possible defects in growth cone guidance. Here we focus on two new genes: commissureless (comm) and roundabout (robo). Mutations in comm lead to an absence of nearly all CNS axon commissures, such that growth cones that normally project across the midline instead now extend only on their own side. Mutations in robo lead to the opposite misrouting, such that some growth cones that normally extend only on their own side instead now project across the midline. The phenotypes of these two genes suggest that they may encode components of attractive and repulsive signaling systems at the midline that either guide growth cones across the midline or keep them on their own side.
Asunto(s)
Axones/fisiología , Sistema Nervioso Central/embriología , Drosophila/genética , Genes de Insecto , Mutación , Animales , Drosophila/embriología , Vías Nerviosas/embriología , FenotipoRESUMEN
Previous studies have led to the suggestion that the midline of the developing Drosophila central nervous system might serve a dual function during growth cone guidance, providing attractive signals for those growth cones that project across the midline, and at the same time repulsive signals for those growth cones that stay on only their own side. In an attempt to discover some of the genes encoding key components of these two guidance systems, we performed a large-scale screen in Drosophila for mutants that affect the development of the axon commissures. Here we review the mutant phenotypes of two new genes in which either too few (commissureless) or too many (roundabout) growth cones cross the midline. These phenotypes suggest that these two genes encode important components for growth cone guidance at the midline.
Asunto(s)
Sistema Nervioso Central/embriología , Drosophila/embriología , Genes de Insecto , Alelos , Animales , Axones/ultraestructura , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Diferenciación Celular , Movimiento Celular , Quimiotaxis , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Drosophila/genética , Proteínas de Drosophila , Embrión no Mamífero/fisiología , Embrión no Mamífero/ultraestructura , Inducción Embrionaria , Saltamontes/embriología , Morfogénesis/genética , Neuronas/ultraestructura , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Fenotipo , Vertebrados/embriología , Vertebrados/genéticaAsunto(s)
Alprostadil/análogos & derivados , Cartílago Articular/efectos de los fármacos , Glicosaminoglicanos/biosíntesis , Salicilatos/farmacología , Alprostadil/farmacología , Animales , Aspirina/farmacología , Cartílago Articular/metabolismo , Células Cultivadas , Medios de Cultivo , Perros , Glicosaminoglicanos/antagonistas & inhibidores , Húmero , Misoprostol , Salicilatos/metabolismo , Ácido Salicílico , Salicilato de Sodio/farmacologíaAsunto(s)
Acetatos , Alprostadil/análogos & derivados , Inmunosupresores , Inhibidores de la Lipooxigenasa , Fenoles , Linfocitos T/efectos de los fármacos , Alprostadil/farmacología , Araquidonato 5-Lipooxigenasa/farmacología , Supervivencia Celular/efectos de los fármacos , Citotoxicidad Inmunológica/efectos de los fármacos , Humanos , Inmunidad Celular/efectos de los fármacos , Técnicas In Vitro , Interleucina-2/biosíntesis , Células Asesinas Naturales/inmunología , Activación de Linfocitos/efectos de los fármacos , Receptores de Interleucina-2/metabolismo , Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Carbetimer is a new antineoplastic agent whose main side effects consist of neurotoxicity and long-term dose-dependent hypercalcemia. We previously showed that Carbetimer is a potent calcium chelator responsible for an acute decrease in ionized Ca levels observed in vivo. However, the mechanism of the progressive increase in serum Ca remains unknown. We have evaluated the bone-resorbing effects of Carbetimer on 45Ca-prelabelled neonatal mouse calvariae. Carbetimer induced a dose-dependent increase in 45Ca release which started at a concentration of 1 mg/ml and reached a mean of 3.3 times the control values at 10 mg/ml. This marked increase in 45Ca release was similar on previously killed bones and could not be inhibited by calcitonin. Such concentrations are probably therapeutically relevant given the known affinity of Carbetimer for bone and the large daily doses administered to cancer patients (10-15 g). Since Carbetimer could exert its antineoplastic action through immunomodulation, we also studied its effects on the production of TNF-alpha and IFN-gamma which are also known to affect bone metabolism. Carbetimer did not stimulate TNF-alpha release from isolated normal human monocytes or lymphocytes, but it markedly inhibited T-lymphocyte production of IFN-gamma, which became undetectable at a concentration of 1 mg of Carbetimer/ml. In summary, Carbetimer-induced hypercalcemia appears to be due to a direct stimulation of osteolysis, but possibly also to an inhibition of IFN-gamma production.