ABSTRACT
An antisense (AS) oligodeoxynucleotide based on a conserved sequence in the three isoforms of the Na(+)/Ca(2+) exchanger (NCX) was used to decrease expression of this Ca(2+) transporter in primary neuronal cultures. Two AS oligo applications decreased NCX activity by approximately 40% within 12-24 h, and neither sense (S) or missense (MS) oligos altered NCX activity. The reduced NCX expression was confirmed by immunoblots and enzyme-linked immunosorbent assays (ELISAs). Resting [Ca(2+)](i) levels were 20% higher in AS-treated neurons and showed a slower return to baseline levels following activation of Ca(2+) influx by N-methyl-D-aspartate (NMDA). These results suggest that NCX plays a significant role in maintaining neuronal Ca(2+) homeostasis and in restoring baseline Ca(2+) levels following depolarization.
Subject(s)
Brain/drug effects , Neurons/drug effects , Oligonucleotides, Antisense/pharmacology , Sodium-Calcium Exchanger/antagonists & inhibitors , Animals , Brain/cytology , Brain/metabolism , Calcium/metabolism , Cells, Cultured , Conserved Sequence/genetics , Enzyme-Linked Immunosorbent Assay , Fluorescent Dyes , Fura-2 , Homeostasis/drug effects , Immunoblotting , Intracellular Fluid/metabolism , N-Methylaspartate/metabolism , N-Methylaspartate/pharmacology , Neurons/cytology , Neurons/metabolism , Oligonucleotides, Antisense/genetics , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/genetics , Rats , Rats, Sprague-Dawley , Sodium-Calcium Exchanger/genetics , Sodium-Calcium Exchanger/metabolismABSTRACT
Slit is secreted by midline glia in Drosophila and functions as a short-range repellent to control midline crossing. Although most Slit stays near the midline, some diffuses laterally, functioning as a long-range chemorepellent. Here we show that a combinatorial code of Robo receptors controls lateral position in the CNS by responding to this presumptive Slit gradient. Medial axons express only Robo, intermediate axons express Robo3 and Robo, while lateral axons express Robo2, Robo3, and Robo. Removal of robo2 or robo3 causes lateral axons to extend medially; ectopic expression of Robo2 or Robo3 on medial axons drives them laterally. Precise topography of longitudinal pathways appears to be controlled by a combination of long-range guidance (the Robo code determining region) and short-range guidance (discrete local cues determining specific location within a region).
Subject(s)
Drosophila Proteins , Drosophila/embryology , Gene Expression Regulation, Developmental , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , Animals , Cell Movement/physiology , Drosophila/physiology , Microscopy, Immunoelectron , Mutagenesis/physiology , Nerve Tissue Proteins/analysis , Nervous System/chemistry , Nervous System/embryology , Neurons/physiology , Neurons/ultrastructure , Phenotype , Receptors, Immunologic/analysis , Roundabout ProteinsABSTRACT
Previous studies showed that Roundabout (Robo) in Drosophila is a repulsive axon guidance receptor that binds to Slit, a repellent secreted by midline glia. In robo mutants, growth cones cross and recross the midline, while, in slit mutants, growth cones enter the midline but fail to leave it. This difference suggests that Slit must have more than one receptor controlling midline guidance. In the absence of Robo, some other Slit receptor ensures that growth cones do not stay at the midline, even though they cross and recross it. Here we show that the Drosophila genome encodes three Robo receptors and that Robo and Robo2 have distinct functions, which together control repulsive axon guidance at the midline. The robo,robo2 double mutant is largely identical to slit.
Subject(s)
Central Nervous System/metabolism , Drosophila Proteins , Nerve Tissue Proteins/metabolism , Receptors, Cell Surface/metabolism , Receptors, Immunologic/metabolism , Animals , Axons/metabolism , COS Cells , Cell Line , Central Nervous System/cytology , Central Nervous System/embryology , Cloning, Molecular , Dimerization , Down-Regulation/drug effects , Down-Regulation/genetics , Drosophila , Gene Expression/drug effects , Growth Cones/pathology , Humans , Insect Proteins/genetics , Insect Proteins/metabolism , Ligands , Membrane Proteins/metabolism , Membrane Proteins/pharmacology , Molecular Sequence Data , Mutagenesis, Insertional , Nerve Tissue Proteins/genetics , Phenotype , RNA, Messenger/biosynthesis , Receptors, Cell Surface/genetics , Receptors, Immunologic/genetics , Roundabout ProteinsABSTRACT
Previous studies suggested that Roundabout (Robo) is a repulsive guidance receptor on growth cones that binds to an unknown midline ligand. Here we present genetic evidence that Slit is the midline Robo ligand; a companion paper presents biochemical evidence that Slit binds Robo. Slit is a large extracellular matrix protein expressed by midline glia. In slit mutants, growth cones enter the midline but never leave it; they abnormally continue to express high levels of Robo while at the midline. slit and robo display dosage-sensitive genetic interactions, indicating that they function in the same pathway. slit is also required for migration of muscle precursors away from the midline. Slit appears to function as a short-range repellent controlling axon crossing of the midline and as a long-range chemorepellent controlling mesoderm migration away from the midline.
Subject(s)
Drosophila Proteins , Drosophila/metabolism , Insect Proteins/metabolism , Nerve Tissue Proteins/metabolism , Receptors, Immunologic/metabolism , Animals , Axons/physiology , Base Sequence , Body Patterning , Cloning, Molecular , DNA, Complementary , Drosophila/embryology , Drosophila/genetics , Gene Expression Regulation , Insect Proteins/genetics , Ligands , Molecular Sequence Data , Mutagenesis , Nerve Tissue Proteins/genetics , Open Reading Frames , Phenotype , Receptors, Immunologic/genetics , Roundabout ProteinsABSTRACT
Extending axons in the developing nervous system are guided in part by repulsive cues. Genetic analysis in Drosophila, reported in a companion to this paper, identifies the Slit protein as a candidate ligand for the repulsive guidance receptor Roundabout (Robo). Here we describe the characterization of three mammalian Slit homologs and show that the Drosophila Slit protein and at least one of the mammalian Slit proteins, Slit2, are proteolytically processed and show specific, high-affinity binding to Robo proteins. Furthermore, recombinant Slit2 can repel embryonic spinal motor axons in cell culture. These results support the hypothesis that Slit proteins have an evolutionarily conserved role in axon guidance as repulsive ligands for Robo receptors.
Subject(s)
Axons/physiology , Conserved Sequence , Drosophila Proteins , Evolution, Molecular , Nerve Tissue Proteins/physiology , Receptors, Immunologic/metabolism , Amino Acid Sequence , Animals , Base Sequence , COS Cells , Cell Line, Transformed , DNA, Complementary , Drosophila , Humans , Insect Proteins/genetics , Insect Proteins/metabolism , Mice , Molecular Sequence Data , Motor Neurons/physiology , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Protein Processing, Post-Translational , Rats , Receptors, Immunologic/genetics , Sequence Homology, Amino Acid , Spinal Cord , Roundabout ProteinsABSTRACT
The midline glia are specialized, nonneuronal cells at the midline of the Drosophila central nervous system (CNS). During development, the midline glia provide guidance cues for extending axons. At the same time, they migrate and help separate the two axon commissures. They then wrap around and ensheath the commissural axons. In many segments, a few of the glia do not enwrap the axons, and these cells die. The wrapper gene encodes a novel member of the immunoglobulin (Ig) superfamily. Wrapper protein is expressed specifically on the surface of midline glia. In wrapper mutant embryos, the midline glia express their normal guidance cues and migrate normally. However, they do not ensheath the commissural axons, and as a result, the glia die. In the absence of Wrapper, the two axon commissures are not properly separated.
Subject(s)
Axons/physiology , Drosophila Proteins , Immunoglobulins/chemistry , Insect Proteins/chemistry , Nerve Tissue Proteins/biosynthesis , Neuroglia/metabolism , Amino Acid Sequence , Animals , Drosophila/embryology , Drosophila/genetics , Gene Expression Regulation, Developmental , Immunoglobulins/genetics , Insect Proteins/genetics , Molecular Sequence Data , Multigene Family , Mutagenesis , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/physiology , Sequence Homology, Amino AcidSubject(s)
Carrier Proteins/antagonists & inhibitors , Carrier Proteins/genetics , Myocardium/metabolism , Neurons/metabolism , Oligonucleotides, Antisense/pharmacology , Animals , Calcium/metabolism , Carrier Proteins/metabolism , Cells, Cultured , Embryo, Mammalian , Neurons/drug effects , Rats , Sodium/metabolism , Sodium-Calcium ExchangerABSTRACT
Antisense oligodeoxynucleotides based on the sequence of the cardiac Na+/Ca2+ exchanger were used to study the effects of decreasing expression of this protein on Na(+)-dependent Ca2+ uptake activity and on the spontaneous beating rate of cultured rat cardiac myocytes. Antisense oligos reduced Na+/Ca2+ exchanger activity of myocytes approximately 30-40%, compared to sense-treated or untreated controls. Antisense treatment also increased the spontaneous beating rate of myocardial cells from an average rate of 40 beats to 69 beats per minute, compared to sense-treated or untreated controls. These results indicate that reduced expression of the Na+/Ca2+ exchanger significantly alters intracellular Ca2+ regulation leading to an enhanced spontaneous beating rate and, presumably, a shortened duration of the cardiac cycle in developing myocytes.
