To establish the identity of the Eph-expressing neurons, transgenic lines were generated carrying the Eph neural enhancer fused to an axon-targeted reporter gene. Eight genomic fragments covering 20 kb were tested for enhancer activity by examining transgenic individuals, each carrying a fragment fused to either tau-myc or tau-lacZ. As judged by reporter gene expression, genomic fragments dekD (for Drosophila Eph kinaseD) through dekH do not demonstrate any enhancer activity. Fragments dekA and dekB drive reporter gene expression in patterns completely unrelated to that of Eph and thus appear to contain enhancers for a different gene located upstream of the Eph locus. In contrast, dekC, a 5.5-kb fragment located 2.2 kb upstream of the putative transcriptional start site of Eph, contains the Eph neural enhancer. In dekC-tau-lacZ embryos, tau-beta-gal expression closely resembles the pattern of Eph expression and is confined to a large subset of interneurons that project axons in the commissures and connectives of the VNC. Similar to the antibody result, no reporter expression could be detected in motor neurons. dekC also drives expression of tau-beta-gal in third-instar larval photoreceptor cells and their axon projections into the optic brain lobes (Scully, 1999).
To test if Drosophila Ephrin is able to bind to axons, the secreted EphrinDeltaC-term truncation, which has an intact receptor binding domain, was expressed. Expression of EphrinDeltaC-term in muscles overlying the CNS (GAL4 line 24B) results in a specific accumulation of the truncated protein on axons. In vertebrates, injection of secreted forms of ephrins give a dominant negative phenotype. However, expression of EphrinDeltaC-term in CNS or muscles (elav-GAL4, sim-GAL4 and GAL424B) failed to cause any obvious defects. This lack of phenotypes is most likely due to insufficient levels of expression (Bossing, 2002).
The accumulation of EphrinDeltaC-term around axons suggests that Ephrin may bind to Eph receptor tyrosine kinase (Eph). The binding between Ephrin and Eph was confirmed in cell culture. Drosophila S2 cells were transfected with a UAS construct encoding the extracellular part of Eph fused to GFP (Ephex-GFP). After incubation with Ephex-GFP-containing medium, non-permeabilized Schneider cells can be labelled with anti-GFP. Hence, Ephex-GFP can bind onto the surface of S2 cells that express endogenous Ephrin. To confirm that Ephex-GFP binds to Ephrin the level of Ephrin expression was lowered by incubation of S2 cells with dsEphrin mRNA. Indeed, Ephrin RNAi treatment of S2 cells diminishes the binding of Ephex-GFP. Control incubation of Schneider cells with dsGFP RNA (GFP RNAi) or dsEph RNA (Eph RNAi) does not interfere with the binding (Bossing, 2002).
VAP proteins (human VAPB/ALS8, Drosophila VAP33, and C. elegans VPR-1) are homologous proteins with an amino-terminal major sperm protein (MSP) domain and a transmembrane domain. The MSP domain is named for its similarity to the C. elegans MSP protein, a sperm-derived hormone that binds to the Eph receptor and induces oocyte maturation. A point mutation (P56S) in the MSP domain of human VAPB is associated with Amyotrophic lateral sclerosis (ALS), but the mechanisms underlying the pathogenesis are poorly understood. This study shows that the MSP domains of VAP proteins are cleaved and secreted ligands for Eph receptors. The P58S mutation in VAP33 leads to a failure to secrete the MSP domain as well as ubiquitination, accumulation of inclusions in the endoplasmic reticulum, and an unfolded protein response. It is proposed that VAP MSP domains are secreted and act as diffusible hormones for Eph receptors. This work provides insight into mechanisms that may impact the pathogenesis of ALS (Tsuda, 2008).
The mechanisms that underlie ALS are poorly understood. ALS is associated with the dysfunction or death of motor neurons in the motor cortex, brain stem, and spinal cord. About 10%-15% of all ALS cases are familial, whereas 85%-90% are sporadic. The most common form of familial ALS is caused by mutations in superoxide dismutase 1 (SOD1). Another gene, ALS8, that has been identified that causes familial ALS. This gene encodes the VAMP (synaptobrevin)-associated protein B (VAPB). Lesions in SOD1 and ALS8 have been shown to cause a wide variety of symptoms that typically include motor neuron death, but vary widely in the age of onset, the speed of progression, and the motor neuron populations that are affected. For example, a single amino acid change in ALS8 (P56S) causes typical ALS, atypical slowly progressive ALS, and spinal muscular atrophy (SMA) with an age of onset between 25 and 52 years and a speed of progression between 2 and 30 years. The cause of this variation may be due to genetic modifiers, partial redundancy, or environment (Tsuda, 2008).
VAPB is closely related to VAPA, which has been shown to associate with the cytoplasmic face of the endoplasmic reticulum (ER) and the Golgi apparatus. Human VAPB (hereafter named hVAP) protein is about 30 kDa and has homologs in C. elegans (VPR-1), Drosophila (VAP33-A, hereafter named dVAP), and numerous other species, including yeast (Scs2p). VAPs consist of an amino (N)-terminal domain of about 125 residues called the major sperm protein (MSP) domain, which is conserved among all VAP family members. The central region is predicted to form a coiled-coil motif. The hydrophobic carboxy (C)-terminus acts as a membrane anchor. The MSP domain is named for its similarity to nematode MSPs, the most abundant proteins in nematode sperm. MSP and VAP MSP domains fold into evolutionarily conserved immunoglobulin-type seven-stranded β sandwiches, suggesting a common function (Tsuda, 2008).
The main difference between VAPs and MSP is their proposed functions. C. elegans MSPs do not contain a coiled-coil motif or a transmembrane domain. MSPs have an intracellular cytoskeletal function, which depends on their ability to polymerize in the absence of actin or myosin and an extracellular signaling function during fertilization. MSP is secreted from the sperm cytosol into the reproductive tract by an unconventional process. Extracellular MSP directly binds to the VAB-1 Eph receptor and other yet-to-be-identified receptors on oocyte and ovarian sheath cell surfaces. MSP induces oocyte maturation, which prepares oocytes for fertilization and embryogenesis, and sheath contraction (Tsuda, 2008 and references therein).
The Eph receptors are an evolutionarily conserved class of receptor tyrosine kinases that bind to membrane-attached ligands called Ephrins. Ephrins act in parallel to gap junctions to inhibit oocyte maturation, and MSP antagonizes this inhibitory circuit. MSP induces activation of the MAP kinase and Ca2+/calmodulin-dependent protein kinase II cascades as well as reorganization of the oocyte microtubule cytoskeleton (Tsuda, 2008 and references therein).
The biological function of VAPs is not well understood. Yeast Scs2p is involved in phosphatidylinositol-4-phosphate synthesis and ceramide transport. VAPs have been reported to associate with the ER. Overexpression of hVAP in human cells affects the structural integrity of the ER through interaction with Nir (N-terminal domain-interacting receptor) proteins. VAPs also interact with oxysterol-binding protein (OSBP) and ceramide transfer protein. These interactions are each mediated through FFAT (two phenylalanines in an acidic tract) domains. Taken together, the results suggest that VAPs might play a role in fatty acid metabolism (Tsuda, 2008 and references therein).
To further define the role of VAPs, Drosophila dVAP has been characterized. dVAP modulates the number and size of neuromuscular junction (NMJ) boutons. Loss of dVAP disrupts the presynaptic microtubule architecture and causes an increase in miniature excitatory junctional potential (mEJP) size as well as an increase in postsynaptic glutamate receptor clustering (Tsuda, 2008).
This study presents evidence that VAP MSP domains are secreted ligands for Eph receptors. It is proposed that secreted MSP domains function as trophic factors by binding to Eph receptors and other cell-surface receptors. The P56S mutation that causes ALS8 (P58S in dVAP) induces insoluble aggregates that are ubiquitinated in flies. The mutation also leads to an accumulation of mutant and wild-type protein in the ER, an unfolded protein response (UPR), and a failure to secrete the MSP domain. Collectively, these results suggest that P56S affects a cell-autonomous pathway involving the ER and UPR as well as a cell nonautonomous pathway involving Eph receptor signaling (Tsuda, 2008).
ALS is a disease caused by death of anterior horn motor neurons in the spinal cord and neurons in motor cortex, after decades of apparently normal development and function. Familial and sporadic ALS cases as well as mouse models induced by overexpressing mutant SOD1 indicate that all forms lead to intracellular cytoplasmic protein inclusions containing ubiquitinated proteins. In flies expressing P58S dVAP, cytoplasmic inclusions and other key characteristics of ALS were found. (1) P58S dVAP protein induces ubiqutinated inclusions. (2) The protein inclusions are associated with the ER and appear to be electron-dense ER expansions. (3) Several key ER proteins colocalize with these inclusions. Finally, mutant dVAP induces a unfolded protein response (UPR). These data show at least three important parallels with ALS and SOD1 mouse models: cytoplasmic inclusions, ubiquitination, and the UPR. The UPR-induced stress caused by P58S dVAP could eventually result in cellular damage or neuronal death (Tsuda, 2008).
Another feature associated with ALS is that the disease may have a cell-non-autonomous component. VAP MSP domains can be secreted, although not all cell types appear capable of secretion in flies. The VAP proteins, including the yeast homolog SCS2, have been proposed to be type II-membrane proteins (Kagiwada, 1998). Since the proteins lack an N-terminal signal sequence, similar to MSP, secretion is likely to occur by an unconventional mechanism as observed for the C.elegans MSP proteins. In addition, the hVAP MSP domain is present in blood serum). The MSP in serum may be able to bind to Eph receptors present on endothelial cells, which regulate angiogenesis. Indeed, SOD1 mutants display defects in the tight junctions between endothelial cells, and endothelial damage occurs prior to motor neuron degeneration. Interestingly, it has recently been reported that VAPB is significantly decreased in the spinal cord of SOD1 mutants and human patients with sporadic ALS. It is therefore possible that reduced signaling by the hVAP MSP domain is a mechanism responsible for some nonautonomous features associated with ALS pathogenesis (Tsuda, 2008).
This study shows that secreted MSP domains bind to Eph receptors on the surfaces of cells. Eph receptors also bind to ligands called Ephrins. MSP domains function in vivo to antagonize Ephrin signaling during oocyte maturation and, possibly, amphid neuron migration. Competition assays are consistent with MSP domains competing with Ephrin for Eph receptor binding. In other processes, including worm-DTC cell migration, ovarian sheath contraction, and fly MB formation, MSP domains seem to be required for Eph receptor signaling. Hence, the relationship between MSP and Ephrin ligands to Eph receptor signaling may depend on the developmental context, as previously observed for Ephrins and Eph receptors in mammals. Multiple Ephrins and Eph receptors including EphA4 and A7 are expressed throughout the adult nervous system and in skeletal muscle of vertebrate species. Eph receptors regulate the survival of cultured spinal cord motor neurons and influence proliferation and apoptosis in the adult mammalian CNS. VAP MSP may play a role in motor neuron survival or muscle function through interactions with Eph receptors (Tsuda, 2008).
Glutamate excitotoxicity is likely to play a role in the pathogenesis of ALS. Three lines of evidence suggest that VAP MSP domains might regulate glutamate receptor signaling. (1) Eph receptors directly associate with NMDA-subtype glutamate receptors and regulate clustering in cultured neurons. (2) Loss of dVAP function or overexpression of P58S in flies is associated with increased glutamate receptor clustering and increased amplitudes of mEJPs at the NMJs. (3) MSP and the VAB-1 Eph receptor regulate NMDA receptor function during worm oocyte maturation (Tsuda, 2008 and references therein).
The following model is proposed for the pathogenesis of ALS8. The P56S hVAP protein accumulates in the ER, while the wild-type protein is functional. In time, the aggregates become more prominent, P56S hVAP becomes ubiquitinated, and functional wild-type proteins become trapped in the inclusions. These protein inclusions initiate a UPR that eventually affects cell viability and lead to a decrease in MSP domain secretion. Impaired secretion decreases signaling by Eph receptors and other receptors. The mutant protein therefore causes two different defects: a cell-autonomous defect in the ER that creates a UPR and a cell non-autonomous defect resulting from reduced secretion of VAP MSP, which may function as an autocrine or paracrine signal. Both defects may synergize to produce the key features of ALS pathology. This model provides testable hypotheses and raises questions to be addressed in the future (Tsuda, 2008).
Motor neuron diseases (MNDs) are progressive neurodegenerative disorders characterized by selective death of motor neurons leading to spasticity, muscle wasting and paralysis. Human VAMP-associated protein B (hVAPB) is the causative gene of a clinically diverse group of MNDs including amyotrophic lateral sclerosis (ALS), atypical ALS and late-onset spinal muscular atrophy. The pathogenic mutation is inherited in a dominant manner. Drosophila VAMP-associated protein of 33 kDa A (DVAP-33A) is the structural homologue of hVAPB and regulates synaptic remodeling by affecting the size and number of boutons at neuromuscular junctions. Associated with these structural alterations are compensatory changes in the physiology and ultrastructure of synapses, which maintain evoked responses within normal boundaries. DVAP-33A and hVAPB are functionally interchangeable and transgenic expression of mutant DVAP-33A in neurons recapitulates major hallmarks of the human diseases including locomotion defects, neuronal death and aggregate formation. Aggregate accumulation is accompanied by a depletion of the endogenous protein from its normal localization. These findings pinpoint to a possible role of hVAPB in synaptic homeostasis and emphasize the relevance of the fly model in elucidating the patho-physiology underlying motor neuron degeneration in humans (Chai, 2008).
hVAPB has been shown to be the causative gene of late-onset autosomal dominant forms of motor neuron disorders, including typical and atypical ALS and late-onset spinal muscular atrophy. The pathogenic mutation predicts a substitution of a Serine for a conserved Proline (P56). One of the hallmarks associated with loss-of-function and neuronal overexpression of DVAP-33A is decreased and increased bouton formation at the NMJ, respectively. Despite this structural alteration, synaptic transmission is maintained within a wt range. At the mechanistic level, muscles respond to a decreased number of boutons and quantal content by upregulating quantal size; conversely muscles compensate an increase in number of boutons and quantal content by downregulating quantal size. Compensatory changes in quantal size during synaptic homeostasis are thought to be determined, largely, by the properties of transmitter receptors. At the Drosophila NMJ, there are two classes of glutamate receptors: one set containing the subunit IIA and another one containing the subunit IIB. In DVAP-33A loss-of-function mutations, the increase in quantal size is associated with an increase in the number and average cluster volume of subunit IIA. Conversely, the decrease in quantal size in the oversprouting mutants is accompanied by a decrease in the level of post-synaptic receptor subunit IIA and a reduction in the average cluster volume for several subunits. In agreement with these data, the IIA subunit receptors have been shown to affect quantal size and receptor channel open time. Similar to the oversprouting mutants, in synapses lacking the receptor subunit IIA, a homeostatic increase in neurotransmitter release compensates for the reduction in quantal size and the evoked response is maintained within normal values. These data indicate that expression levels of VAP proteins play a crucial role in synaptic homeostasis by coordinating structural remodeling and post-synaptic sensitivity to neurotransmitter to ensure synaptic efficacy (Chai, 2008).
Interestingly, expression of hVAPB in neurons rescues lethality, morphological and electrophysiological phenotypes associated with DVAP-33A loss-of-function mutations. Moreover, neuronal expression of hVAPB in a wt background induces phenotypes similar to the overexpression of DVAP-33A. These data clearly indicate that DVAP-33A and hVAPB perform homologous functions at the synapse and as a consequence, information gained by studying DVAP-33A is expected to be relevant for hVAPB function as well. Surprisingly, neuronal expression of mutant VAP proteins also rescues all phenotypes associated with mutations in DVAP-33A. Two alternative scenarios could be proposed to explain these data: the mutation is irrelevant for the ALS8 pathogenesis or the mutant allele has a pathogenic effect while retaining certain functional properties of the wt protein. The second hypothesis is favored for the following reasons. (1) The P56S mutation in hVAPB has been reported to be causative for an inherited form of MNDs in humans. This mutation affects nine related families totaling 1500 individuals of which 200 suffer from motor neuron disorders. (2) A genetic model for MNDs was generated where the expression of the aberrant VAP recapitulates major hallmarks of the human disease, clearly indicating that the mutation has a pathogenic effect. (3) The data suggest that both the Drosophila and the human mutant proteins retain some functional wt properties such as the ability to self-oligomerize. However, neuronal expression of the pathogenic protein induces aggregate formation and depletes the wt protein from its normal localization. These effects are not observed when the wt protein is overexpressed, suggesting that the mutant protein has acquired a new, potentially toxic property (Chai, 2008).
Indeed, one of the most common features of MNDs and nearly all neurodegenerative diseases is the accumulation of aggregates that are intensively immuno-reactive to disease-related proteins. Each disease, however, differs with respect to the anatomical location and morphology of the aggregates. The major component of the aggregates is usually the protein encoded by the gene mutated in the familial forms, which is also unique to each disease. Despite this diversity, a bulk of circumstantial evidence support the hypothesis that aggregates are typical hallmarks of neurodegenerative diseases and have a toxic effect on neurons. While no autopsy material is available for familial cases with the P56S mutation, SOD1-positive inclusions have been reported in human sporadic and familial ALS cases as well as in SOD1 mouse models. This study found the presence of aggregates that are intensively immuno-reactive for DVAP-33A both in neuronal cell bodies and in nerve fibers of the MND model. Interestingly, hVAPB carrying the pathogenic mutation has also been shown to undergo intracellular aggregation when expressed in a cell culture system. However, similarities between human disease and the fly model are not limited to aggregate formation as flies expressing transgenic VAP proteins carrying the ALS8 mutation, exhibit other hallmarks of the human disease such as neuronal cell death, muscle wasting and defective locomotion behavior (Chai, 2008).
Although it remains to be established whether the VAP protein in the aggregates represents the mutant protein, the endogenous protein or a mixture of both, a regional decrease in the level of the endogenous protein is clearly observed. The DVAP-33A protein that is normally associated with the plasma membrane in neuronal cell bodies and at the neuromuscular synapses is nearly undetectable in DVAPP58S transgenic animals. As a consequence of the decrease in synaptic levels of the endogenous protein, a decrease in the number of boutons is observed. It has been previously shown that DVAP-33A regulates bouton formation at the synapse in a dosage-dependent manner. Despite these structural alterations a homeostatic mechanism is established to maintain synaptic efficacy within functional boundaries. It is speculated that the depletion of the endogenous protein from its normal localization and the formation of aggregates would affect the homeostatic mechanism linking structural remodeling and synaptic efficacy controlled by DVAP-33A. Although not directly tested in this model, experiments in cell culture show that overexpression of mutant hVAPB induces formation of aggregates in which the endogenous wt protein is recruited. This would suggest that the pathogenic allele functions as a dominant negative. However, the depletion of the endogenous protein from its normal localization cannot be the principal mechanism of the disease as mutants lacking DVAP-33A do not develop MND. It is therefore possible that the pathogenic allele has acquired an abnormal, new toxic activity. Similar to what has been proposed for other neurodegenerative diseases, the formation of aggregates may directly interfere with critical cellular processes and/or compromise the ability of the system to keep up with the degradation of aggregated proteins (Chai, 2008).
Taken together these data offer experimental support to the hypothesis that VAP proteins play a conserved role in synaptic homeostasis and emphasize the relevance of this fly model in fostering an understanding of the molecular mechanisms underlying VAP-induced motor neuron degeneration in humans (Chai, 2008).
In situ hybridization to whole-mount embryos reveals that Eph transcripts are present in precellular blastoderm stage embryos, as well as in unfertilized eggs, demonstrating that EPH mRNA is maternally supplied. A higher concentration of Eph transcripts is evident in the posterior pole of the embryo. In the embryo, zygotic transcription of Eph is confined to the nervous system. Expression commences in a large subset of neurons within the brain and ventral nerve cord (VNC) at stage 13 when neurons begin elongating axons. Eph continues to be expressed in the larval CNS and imaginal discs, as well as pupal and adult stages as assayed by Northern blot analysis. To further define which neurons express Eph, antibodies were generated to the cytoplasmic portion of the Eph protein. Immunostaining with an affinity-purified mouse antibody reveals that Eph is highly targeted to axons and growth cones of developing neurons within the VNC. Highest levels are present on axons within the connectives; lower levels are detectable in the commissures. Based upon the numbers and morphology of the staining axons, Eph is expressed by a large subset of interneurons and does not appear to be expressed by motor neurons (Scully, 1999).
Roles for Eph receptor tyrosine kinase signaling in the formation of topographic patterns of axonal connectivity have been well established in vertebrate visual systems. A role for a Drosophila Eph receptor tyrosine kinase (Eph) in the control of photoreceptor axon and cortical axon topography in the developing visual system is described. Although uniform across the developing eye, Eph is expressed in a concentration gradient appropriate for conveying positional information during cortical axon guidance in the second-order optic ganglion, the medulla. Disruption of this graded pattern of Eph activity by double-stranded RNA interference or by ectopic expression of wild-type or dominant-negative transgenes perturbs the establishment of medulla cortical axon topography. In addition, abnormal midline fasciculation of photoreceptor axons results from the eye-specific expression of the dominant-negative Eph transgene. These observations reveal a conserved role for Eph kinases as determinants of topographic map formation in vertebrates and invertebrates (Dearborn, 2002).
EPH expression coincides spatially and temporally with the differentiation and outgrowth of photoreceptor and cortical cell axons in the developing eye and optic ganglia, respectively. Eph antigen accumulates on the axons and growth cones of these neurons. Interestingly, the level of Eph immunoreactivity varies in a position-specific manner within each tissue. As photoreceptor axons grow into the lamina, Eph antigen is most strongly concentrated on the older photoreceptor growth cones that terminate at the posterior of the lamina. Eph antigen is also most strongly concentrated in the prospective posterior medulla neuropil that contains the axons of the earliest differentiating cortical neurons and R7-R8 photoreceptors. One might suppose that this distribution of antigen reflects the accumulation of Eph with time after the onset of differentiation. However, the observation that the anteroposterior gradient on these axons and growth cones persists into the early pupal stage suggests that it reflects spatially distinct expression or stability of Eph. Eph also displays a symmetrical concentration gradient on the dorsoventral axis of the medulla. Cortical neurons at the prospective midline of the medulla express the highest levels of Eph. In analogy with vertebrate Eph family members, the position-specific distribution of Drosophila Eph might reflect a role in the guidance of cortical cell axons to correct topographic positions. Consistent with this model, the single ephrin-like molecule encoded in the Drosophila genome (see Ephrin) is expressed in a gradient pattern that is complimentary to the Eph dorsoventral pattern in the medulla. The centripetal trajectories of cortical cell axons might thus rely on a repulsive interaction between Eph-bearing midline growth cones and a dorsoventral localized ephrin ligand. The apparently uniform expression of Eph on the dorsoventral axis of the eye does not preclude a role in the dorsoventral guidance of photoreceptor axons. In the chick, the response of retinal growth cones to target-derived ephrin can be modulated by nonuniform coexpression of an ephrin ligand by retinal ganglion neurons (Dearborn, 2002).
To gain insight into the role of Eph in the establishment of topographic connectivity, double-stranded RNA interference was used to reduce or eliminate Eph expression. eph dsRNA was injected into syncytial stage embryos to perturb eph expression at the larval time points relevant to axon targeting in the adult visual system. The reliability of RNAi was enhanced by using unique regions of eph as dsRNA template and by carefully determining the level of Eph antigen in the visual systems of dsRNA-injected animals. The data reveal that defects in photoreceptor and medulla cortical axon projections are associated with the loss of Eph expression. In the 20% of specimens that display a significant reduction or complete loss of Eph expression, the eye and medulla cortex formed with apparently normal size and cellular organization. The severe defects in medulla neuropil topography observed were most consistent with mistargeting of cortical axons. Given the severity of these defects in this target destination for the R7-R8 photoreceptor axons, it cannot be concluded that loss of Eph expression affected photoreceptor axons directly. The low penetrance of dsRNA-mediated effects (~20%) is consistent with previous reports on the effects of embryonic introduction of dsRNA on postembryonic and adult gene expression. Thus, RNAi-mediated reduction or elimination of Eph expression indicates that Eph is required for normal optic ganglia formation (Dearborn, 2002).
This conclusion was supported and refined by examining the consequences of expressing wild-type (UAS-eph+) and dominant-negative (UAS-ephDN) transgenes in the visual system. In the developing eye, transgene expression was driven in differentiating ommatidial cell clusters with ey-GAL4 and GMR-GAL4. Photoreceptor axon fascicles from each ommatidial unit (R1-R8) are normally bundled together as they traverse the optic stalk and then separate on the dorsoventral axis as they turn toward retinotopic destinations in the lamina field. With the expression of ephDN, the photoreceptor axon fascicles located near the midline were affected at the entrance into the lamina, in which they remained bundled together and often projected out of the lamina field. Axons of dorsally and ventrally located photoreceptors projected to topographically appropriate locations, despite their expression of ephDN. These defects were also observed when the FLP-out GAL4 driver was used to express ephDN in clones restricted to the developing eye. These observations are at odds with those of Scully (1999), who reported GMR-GAL4-driven expression of a putative dominant-negative eph construct did not cause defects in photoreceptor axon pathfinding. However, their construct was made by introducing a single amino acid substitution into the Eph kinase domain to eliminate kinase activity. It is possible that the fasciculation phenotype observed does not require kinase activity but relies on signaling from other Eph intracellular domains that are deleted in the ephDN construct. These observations are consistent with the idea that repulsion mediated by Eph activity is required to separate the axon fascicles as they emerge from the optic stalk. Endogenously truncated isoforms of vertebrate Eph RTKs have been found to promote adhesive interactions when coexpressed with full-length receptors in vitro (Dearborn, 2002).
The possibility that the dorsoventral gradient of Eph expression is necessary for the establishment of medulla cortical axon topography was examined by expressing the eph+ and ephDN transgenes in specific cortical cell populations. The omb-GAL4 driver was used to express the eph+ transgene in dorsally and ventrally located cortical cell populations that normally express little Eph, thus disrupting the Eph gradient on this axis. This resulted in the disruption of the projections of dorsal and ventral cortical cells. Similarly, when an ap-GAL4 driver was used to misexpress eph+ in a subset of cortical cells distributed along the dorsoventral axis, only those cells located in dorsal and ventral locations displayed axon projection defects. Although the omb-GAL4 driver would also yield eph+ expression at the dorsal and ventral margins of the eye and in a subset of optic lobe glia, the similar outcome resulting with ap-GAL4-driven expression (which is not expressed in either of those cell populations) indicates that cortical cell expression of eph+ underlies the axon projection defects. In contrast, ap-GAL4-driven expression of ephDN results in cortical cell axon projection defects at the midline, in which cells normally express the highest levels of Eph. These results are consistent with an interpretation that the requirement for Eph activity is highest at the midline, which coincides with the distribution of Eph along this axis. These observations are also consistent with the activity of the putative ephrin as a growth cone repellent for Eph-positive axons. This ephrin transcript is expressed in a pattern that is complimentary to the Eph pattern on the dorsoventral axis (Dai and Kunes, unpublished observations reported in Dearborn, 2002). More restricted, mosaic expression of the ephDN transgene in both eye and brain tissues using the FLP-out GAL4 driver further confirms a role for Eph in the formation of both retinotopic and cortical cell topographic projections and suggests that relative levels of Eph activity are critical to the establishment of medulla axon topography, observations consistent with studies performed in the mouse (Dearborn, 2002).
In summary, disruption of wild-type Eph expression and/or activity in both photoreceptor and medulla cortical cells results in defects in the axon projections of these cell types consistent with a position-dependent requirement for Eph signaling. These observations provide the first evidence that the underlying mechanisms directing axons to topographically appropriate sites within the brain during visual system development are conserved in vertebrates and invertebrates, relying on position-specific levels of Eph signaling (Dearborn, 2002).
Both Ephrin and Eph map to the fourth chromosome, for which it is very difficult to obtain and maintain mutants by classical genetic techniques. For this reason RNAi was used to inhibit expression. RNAi has rapidly become an accepted technique for generating mutant phenotypes. In test injections of dsEphrin RNA only two out of nine injected embryos show a nearly complete loss of Ephrin, while the remainder retains about 20%-50% of wild-type expression. Therefore, it was not expected that Ephrin RNAi would lead to a mutant phenotype in all injected embryos nor that all segments per embryo would be affected. Indeed, only 65% (13/20) of embryos injected with dsEphrin showed an aberrant phenotype and in total 39% (77/200) of all segments are affected. In four injected embryos all segments were affected. The phenotypes include fused commissures, loss of commissures and breaks in the connectives. Although Ephrin RNAi impedes commissure formation, it does not interfere with the differentiation of midline glia. Injection with dsCFP or buffer does not reduce Ephrin expression but occasionally results in phenotypes similar to dsEphrin injections. However, only 30% (5/15) of dsCFP injected embryos and 23% (4/17) of buffer injected embryos show a phenotype. The number of affected segments is reduced to 6% (9/148, dsCFP) or 8% (13/167, buffer) (Bossing, 2002).
Eph RNAi also results in fused commissures, loss of commissures and breaks in the connectives. The phenotype of Eph RNAi is more severe than for Ephrin RNAi. 80% (12/15) of all embryos had a phenotype and in total 69% (98/142) of all segments were affected. In five embryos all segments were abnormal. The difference in the strength of phenotype could either indicate that additional ligands besides Ephrin signal through Eph or the difference might be caused by the efficiency of RNAi, which varies between different genes (Bossing, 2002).
RNAi against Ephrin and Eph results in the fusion or loss of commissures and breaks in the connectives. Using a general axon marker, the origin of these phenotypes is not clear. Therefore the behaviour of single axons was followed in RNAi-treated embryos. The Gal4 line CY27 primarily drives expression of UAS-taumGFP6 in 2 interneurons per hemisegment, the vMP2 and dMP2 neuron. The MP2 neurons are among the first neurons to extend their axons along the connectives. In differentiated embryos the projections of these neurons form a tight fascicle which extends close and in parallel to the midline. Loss of Ephrin or Eph causes the axons of the MP2 neurons to project aberrantly out of the CNS. In 75% of embryos (15/20, Ephrin RNAi) and 82% of embryos (14/17, Eph RNAi), MP2 axons exiting the CNS were found. In the GAL4 line CY27, additional interneurons (i.e., UMI neurons) start to express GFP in late embryogenesis. No attempt was made to examine these weak projections in detail but it was noticed that many of these interneuronal axons also project out of the CNS. Therefore, signalling between Ephrin and Eph plays a role in confining interneuronal axons to the connectives (Bossing, 2002).
In vertebrates activation of Eph receptors in axonal growth cones is able to repel axons. It was speculated that despite the structural differences between vertebrate ephrins and Ephrin, the repulsive ability of Ephrin/Eph signalling might be conserved. Ephrin expression along the outer edge of the connectives and between the commissures could create barriers preventing axon extension. Absence of these barriers would be expected to result in fusion of commissures and the exit of interneuronal axons from the CNS, as was observed in RNAi experiments. To test whether Ephrin can act as an axonal repellent, Ephrin was ectopically expressed (Bossing, 2002).
Only 4-6 out of about 20 midline neurons express Ephrin. Ectopic expression of Ephrin in all midline cells (sim-GAL4) causes fusion, severe thinning or loss of commissures without affecting midline glial cell differentiation. Single cell labelling of neural precursors reveals that ectopic Ephrin in midline cells is able to prevent the midline crossing of axons. In all clones with contralateral axons, the axons are stalled at the midline. Ectopic midline Ephrin does not affect the extension of ipsilateral axons immediately adjacent to the midline or the determination of midline neurons (judged by the expression of Engrailed, Futsch and Odd-skipped) (Bossing, 2002).
Axonal repulsion by Slit, secreted from midline cells, is one of the major mechanisms controlling axons crossing the midline. It is possible that Ephrin expression at the midline exerts its repulsive effect by upregulating the expression or secretion of Slit. To test if repulsion by Ephrin depends on Slit, Ephrin was expressed ectopically in the midline of embryos mutant for Slit and Robo1, one of the receptors for Slit. Expression of Ephrin in slit/robo double mutants forces axons out of the midline. Therefore, Ephrin/Eph signalling at the ventral midline can act independently of Slit and Robo1 (Bossing, 2002).
Ephrin is expressed in nearly all neurons but not in the longitudinal glia that enwrap the connectives. Ephrin-expressing longitudinal glia were generated by injecting UAS-Ephrin plasmids into the syncytial blastoderm of GAL4MZ1580 embryos. When longitudinal glial cells express Ephrin, breaks are observed in the connectives. The breaks are always located near the glial cell. No breaks are observed when neurons express Ephrin. GFP-expressing longitudinal glial cells also do not disrupt axon extension (UAS-tau-mGFP6 plasmid). In summary, ectopic expression of Ephrin blocks axon extension (Bossing, 2002).
Activation of Eph on axons may be the reason that axons stall at Ephrin-expressing midline cells. In that case lowering the level of Eph activation by reducing Eph expression might allow these axons to overcome this repulsion and restore the commissures. To test this hypothesis Eph expression was lowered by Eph RNAi. Embryos with ectopic midline expression of Ephrin show a strong phenotype. Only 2% (2/100) of all segments have wild-type commissures and embryos are never found in which all segments have normal commissures. Injection of dsEph RNA rescues the commissures. In 30% (8/27) of all injected embryos all segments were restored to wild type. In contrast, in all embryos injected with buffer or dsGFP, segments are found with fused or absent commissures, indicating that ectopic Ephrin is still able to repel axons. In dsEph-injected embryos 33% of all segments have wild-type commissures, whereas in control-injected embryos even fewer segments show normal commissures (Bossing, 2002).
Presumably, it is possible to rescue the commissures with Eph RNAi because dsEph-injected embryos do not always show a loss or fusion of commissures. 20% of injected embryos and 31% of all segments have no phenotype. In the rescued embryos, Eph expression might be lowered enough to overcome the repulsion by ectopic midline Ephrin but not low enough to result in fused or lost commissures (Bossing, 2002).
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