Analysis of gene expression during neurite outgrowth and regeneration.

BACKGROUND: The ability of a neuron to regenerate functional connections after injury is influenced by both its intrinsic state and also by extrinsic cues in its surroundings. Investigations of the transcriptional changes undergone by neurons during in vivo models of injury and regeneration have revealed many transcripts associated with these processes. Because of the complex milieu of interactions in vivo, these results include not only expression changes directly related to regenerative outgrowth and but also unrelated responses to surrounding cells and signals. In vitro models of neurite outgrowth provide a means to study the intrinsic transcriptional patterns of neurite outgrowth in the absence of extensive extrinsic cues from nearby cells and tissues. RESULTS: We have undertaken a genome-wide study of transcriptional activity in embryonic superior cervical ganglia (SCG) and dorsal root ganglia (DRG) during a time course of neurite outgrowth in vitro. Gene expression observed in these models likely includes both developmental gene expression patterns and regenerative responses to axotomy, which occurs as the result of tissue dissection. Comparison across both models revealed many genes with similar gene expression patterns during neurite outgrowth. These patterns were minimally affected by exposure to the potent inhibitory cue Semaphorin3A, indicating that this extrinsic cue does not exert major effects at the level of nuclear transcription. We also compared our data to several published studies of DRG and SCG gene expression in animal models of regeneration, and found the expression of a large number of genes in common between neurite outgrowth in vitro and regeneration in vivo. CONCLUSION: Many gene expression changes undergone by SCG and DRG during in vitro outgrowth are shared between these two tissue types and in common with in vivo regeneration models. This suggests that the genes identified in this in vitro study may represent new candidates worthy of further study for potential roles in the therapeutic regrowth of neuronal connections.

Heterogeneity in synaptic transmission along a Drosophila larval motor axon.

At the Drosophila melanogaster larval neuromuscular junction (NMJ), a motor neuron releases glutamate from 30-100 boutons onto the muscle it innervates. How transmission strength is distributed among the boutons of the NMJ is unknown. To address this, we created synapcam, a version of the Ca2+ reporter Cameleon. Synapcam localizes to the postsynaptic terminal and selectively reports Ca2+ influx through glutamate receptors (GluRs) with single-impulse and single-bouton resolution. GluR-based Ca2+ signals were uniform within a given connection (that is, a given bouton/postsynaptic terminal pair) but differed considerably among connections of an NMJ. A steep gradient of transmission strength was observed along axonal branches, from weak proximal connections to strong distal ones. Presynaptic imaging showed a matching axonal gradient, with higher Ca2+ influx and exocytosis at distal boutons. The results suggest that transmission strength is mainly determined presynaptically at the level of individual boutons, possibly by one or more factors existing in a gradient.

Cross GTPase-activating protein (CrossGAP)/Vilse links the Roundabout receptor to Rac to regulate midline repulsion.

The regulators of the Rho-family GTPases, GTPase-activating proteins (GAPs) and guanine exchange factors (GEFs), play important roles in axon guidance. By means of a functional genomic study of the Rho-family GEFs and GAPs in Drosophila, we have identified a Rho-family GAP, CrossGAP (CrGAP), which is involved in Roundabout (Robo) receptor-mediated repulsive axon guidance. CrGAP physically associates with the Robo receptor. Too much or too little CrGAP activity leads to defects in Robo-mediated repulsion at the midline choice point. The CrGAP gain-of-function phenotype mimics the loss-of-function phenotypes of both Robo and Rac. Dosage-sensitive genetic interactions among CrGAP, Robo, and Rac support a model in which CrGAP transduces signals downstream of Robo receptor to regulate Rac-dependent cytoskeletal changes.

Conserved roles for Slit and Robo proteins in midline commissural axon guidance.

In Drosophila, Slit at the midline activates Robo receptors on commissural axons, thereby repelling them out of the midline into distinct longitudinal tracts on the contralateral side of the central nervous system. In the vertebrate spinal cord, Robo1 and Robo2 are expressed by commissural neurons, whereas all three Slit homologs are expressed at the ventral midline. Previous analysis of Slit1;Slit2 double mutant spinal cords failed to reveal a defect in commissural axon guidance. We report here that when all six Slit alleles are removed, many commissural axons fail to leave the midline, while others recross it. In addition, Robo1 and Robo2 single mutants show guidance defects that reveal a role for these two receptors in guiding commissural axons to different positions within the ventral and lateral funiculi. These results demonstrate a key role for Slit/Robo signaling in midline commissural axon guidance in vertebrates.

Highwire regulates presynaptic BMP signaling essential for synaptic growth.

Highwire (Hiw), a putative RING finger E3 ubiquitin ligase, negatively regulates synaptic growth at the neuromuscular junction (NMJ) in Drosophila. hiw mutants have dramatically larger synaptic size and increased numbers of synaptic boutons. Here we show that Hiw binds to the Smad protein Medea (Med). Med is part of a presynaptic bone morphogenetic protein (BMP) signaling cascade consisting of three receptor subunits, Wit, Tkv, and Sax, in addition to the Smad transcription factor Mad. When compared to wild-type, mutants of BMP signaling components have smaller NMJ size, reduced neurotransmitter release, and aberrant synaptic ultrastructure. BMP signaling mutants suppress the excessive synaptic growth in hiw mutants. Activation of BMP signaling, which in wild-type does not cause additional growth, in hiw mutants does lead to further synaptic expansion. These results reveal a balance between positive BMP signaling and negative regulation by Highwire, governing the growth of neuromuscular synapses.

A genetic screen for synaptic transmission mutants mapping to the right arm of chromosome 3 in Drosophila.

Neuronal function depends upon the proper formation of synaptic connections and rapid communication at these sites, primarily through the regulated exocytosis of chemical neurotransmitters. Recent biochemical and genomic studies have identified a large number of candidate molecules that may function in these processes. To complement these studies, we are pursuing a genetic approach to identify genes affecting synaptic transmission in the Drosophila visual system. Our screening approach involves a recently described genetic method allowing efficient production of mosaic flies whose eyes are entirely homozygous for a mutagenized chromosome arm. From a screen of 42,500 mutagenized flies, 32 mutations on chromosome 3R that confer synaptic transmission defects in the visual system were recovered. These mutations represent 14 complementation groups, of which at least 9 also appear to perform functional roles outside of the eye. Three of these complementation groups disrupt photoreceptor axonal projection, whereas the remaining complementation groups confer presynaptic defects in synaptic transmission without detectably altering photoreceptor structure. Mapping and complementation testing with candidate mutations revealed new alleles of the neuronal fate determinant svp and the synaptic vesicle trafficking component lap among the collection of mutants recovered in this screen. Given the tools available for investigation of synaptic function in Drosophila, these mutants represent a valuable resource for future analysis of synapse development and function.

The BMP homolog Gbb provides a retrograde signal that regulates synaptic growth at the Drosophila neuromuscular junction.

We show that the BMP ortholog Gbb can signal by a retrograde mechanism to regulate synapse growth of the Drosophila neuromuscular junction (NMJ). gbb mutants have a reduced NMJ synapse size, decreased neurotransmitter release, and aberrant presynaptic ultrastructure. These defects are similar to those we observe in mutants of BMP receptors and Smad transcription factors. However, whereas these BMP receptors and signaling components are required in the presynaptic motoneuron, Gbb expression is required in large part in postsynaptic muscles; gbb expression in muscle rescues key aspects of the gbb mutant phenotype. Consistent with this notion, we find that blocking retrograde axonal transport by overexpression of dominant-negative p150/Glued in neurons inhibits BMP signaling in motoneurons. These experiments reveal that a muscle-derived BMP retrograde signal participates in coordinating neuromuscular synapse development and growth.

Retrograde control of synaptic transmission by postsynaptic CaMKII at the Drosophila neuromuscular junction.

Retrograde signaling plays an important role in synaptic homeostasis, growth, and plasticity. A retrograde signal at the neuromuscular junction (NMJ) of Drosophila controls the homeostasis of neurotransmitter release. Here, we show that this retrograde signal is regulated by the postsynaptic activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII). Reducing CaMKII activity in muscles enhances the signal and increases neurotransmitter release, while constitutive activation of CaMKII in muscles inhibits the signal and decreases neurotransmitter release. Postsynaptic inhibition of CaMKII increases the number of presynaptic, vesicle-associated T bars at the active zones. Consistently, we show that glutamate receptor mutants also have a higher number of T bars; this increase is suppressed by postsynaptic activation of CaMKII. Furthermore, we demonstrate that presynaptic BMP receptor wishful thinking is required for the retrograde signal to function. Our results indicate that CaMKII plays a key role in the retrograde control of homeostasis of synaptic transmission at the NMJ of Drosophila.

Bi-directional signaling by Semaphorin 1a during central synapse formation in Drosophila.

Semaphorins have been intensively studied for their role in dendritic and axonal pathfinding, but less is known about their potential role in synapse formation. In the adult giant fiber (GF) system of fruit flies (Drosophila melanogaster), we show that transmembrane Semaphorin 1a (Sema-1a) is involved in synapse formation in addition to its role in guidance during pathfinding. Cell-autonomous rescue experiments showed that Sema-1a is involved in assembly of a central synapse and that it is required both pre- and postsynaptically. We also found that pre- but not postsynaptic gain-of-function Sema-1a was able to disrupt the GF-motor neuron synapse and that the phenotype depended on a proline-rich intracellular domain that contains a putative Enabled binding site. We suggest that transmembrane Sema-1a is part of a bi-directional signaling system that leads to the formation of the GF synapse and possibly acts as both a ligand and a receptor.

Genomewide analysis of the Drosophila tetraspanins reveals a subset with similar function in the formation of the embryonic synapse.

Tetraspanins encode a large conserved family of proteins that span the membrane four times and are expressed in a variety of eukaryotic tissues. They are part of membrane complexes that are involved in such diverse processes as intracellular signaling, cellular motility, metastasis, and tumor suppression. The single fly tetraspanin characterized to date, late bloomer (lbm), is expressed on the axons, terminal arbors, and growth cones of motoneurons. In embryos lacking Lbm protein, motoneurons reach their muscle targets, but initially fail to form synaptic terminals. During larval stages, however, functional contacts are formed. The newly available genomic sequence of Drosophila melanogaster indicates the existence of 34 additional members of the tetraspanin family in the fly. To address the possibility that other tetraspanins with functions that might compensate for a lack of lbm exist, we determined the expression domains of the Drosophila tetraspanin gene family members by RNA in situ analysis. We found two other tetraspanins also expressed in motoneurons and subsequently generated a small chromosomal deletion that removes all three motoneuron-specific tetraspanins. The deletion results in a significant enhancement in the lbm phenotype, indicating that the two additional motoneuron-expressed tetraspanins can, at least in part, compensate for the absence of lbm during the formation of the embryonic synapse.

Ectopic expression in the giant fiber system of Drosophila reveals distinct roles for roundabout (Robo), Robo2, and Robo3 in dendritic guidance and synaptic connectivity.

The Roundabout (Robo) receptors have been intensively studied for their role in regulating axon guidance in the embryonic nervous system, whereas a role in dendritic guidance has not been explored. In the adult giant fiber system of Drosophila, we have revealed that ectopic Robo expression can regulate the growth and guidance of specific motor neuron dendrites, whereas Robo2 and Robo3 have no effect. We also show that the effect of Robo on dendritic guidance can be suppressed by Commissureless coexpression. Although we confirmed a role for all three Robo receptors in giant fiber axon guidance, the strong axon guidance alterations caused by overexpression of Robo2 or Robo3 have no effect on synaptic connectivity. In contrast, Robo overexpression in the giant fiber seems to directly interfere with synaptic function. We conclude that axon guidance, dendritic guidance, and synaptogenesis are separable processes and that the different Robo family members affect them distinctly.

wishful thinking encodes a BMP type II receptor that regulates synaptic growth in Drosophila.

We conducted a large-scale screen for Drosophila mutants that have structural abnormalities of the larval neuromuscular junction (NMJ). We recovered mutations in wishful thinking (wit), a gene that positively regulates synaptic growth. wit encodes a BMP type II receptor. In wit mutant larvae, the size of the NMJs is greatly reduced relative to the size of the muscles. wit NMJs have reduced evoked excitatory junctional potentials, decreased levels of the synaptic cell adhesion molecule Fasciclin II, and synaptic membrane detachment at active zones. Wit is expressed by a subset of neurons, including motoneurons. The NMJ phenotype is specifically rescued by transgenic expression of Wit only in motoneurons. Thus, Wit appears to function as a presynaptic receptor that regulates synaptic size at the Drosophila NMJ.

Regulation of cortical dendrite development by Slit-Robo interactions.

Slit proteins have previously been shown to regulate axon guidance, branching, and neural migration. Here we report that, in addition to acting as a chemorepellant for cortical axons, Slit1 regulates dendritic development. Slit1 is expressed in the developing cortex, and exposure to Slit1 leads to increased dendritic growth and branching. Conversely, inhibition of Slit-Robo interactions by Robo-Fc fusion proteins or by a dominant-negative Robo attenuates dendritic branching. Stimulation of neurons transfected with a Met-Robo chimeric receptor with Hepatocyte growth factor leads to a robust induction of dendritic growth and branching, suggesting that Robo-mediated signaling is sufficient to induce dendritic remodeling. These experiments indicate that Slit-Robo interactions may exert a significant influence over the specification of cortical neuron morphology by regulating both axon guidance and dendritic patterning.

Slit1 and Slit2 cooperate to prevent premature midline crossing of retinal axons in the mouse visual system.

During development, retinal ganglion cell (RGC) axons either cross or avoid the midline at the optic chiasm. In Drosophila, the Slit protein regulates midline axon crossing through repulsion. To determine the role of Slit proteins in RGC axon guidance, we disrupted Slit1 and Slit2, two of three known mouse Slit genes. Mice defective in either gene alone exhibited few RGC axon guidance defects, but in double mutant mice a large additional chiasm developed anterior to the true chiasm, many retinal axons projected into the contralateral optic nerve, and some extended ectopically-dorsal and lateral to the chiasm. Our results indicate that Slit proteins repel retinal axons in vivo and cooperate to establish a corridor through which the axons are channeled, thereby helping define the site in the ventral diencephalon where the optic chiasm forms.