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).
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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