Netrin-A and Netrin-B

DEVELOPMENTAL BIOLOGY

Embryonic

See the embryonic expression patterns of NetA and NetB at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.

The first expression of the two genes is observed at the cellular blastoderm stage and is restricted to the presumptive mesoderm. This expression persists through gastrulation and then fades. In the visceral mesoderm, NetA continues to be expressed very strongly, and NetB weakly. Expression of NetB also remains in small patches within the somatic mesoderm layer. From stage 12 onward patches of ectodermal cells that are likely primordia for the tracheal system express NetA. Cells in the dorsal vessel and of the stomatogastric nervous system express NETB. During stage 14, accumulation of NETB is apparent in imaginal disc primordia, including cells that will give rise to the eye-antennal, labial, wing, haltere, and genital discs. Muscles from both the dorsal and ventral muscle groups express NetB. NetA is expressed by dorsal muscles 1 and 2 and is also expressed in a dorsolateral stripe in the epidermis (Harris, 1996, Mitchell, 1996).

Both NetA and NetB are strongly expressed by midline cells during the initial period of commissure formation and axonogenesis in the ventral nerve cord. NetA is initially expressed at stages 12 and 13 by the two anterior pair of midline glia (MGA and MGM) and by the VUM neurons. The expression in the VUM cluster subsequently fades, while the anterior and middle pairs of midline glia continue to express NetA strongly throughout embryogenesis. In addition, a large pair of cells located posterior to the posterior commissure also stains strongly with NetA at this stage. These cells may be associated with the median neuroblast. In contrast with the wide expression of NETA at the midline, NetB is expressed in a more restricted pattern. The midline glia express NETB very strongly, but there is no evidence of strong expression in either the VUM cluster or the MNB cluster. NetB is expressed by many more neurons than NetA. In the peripheral nervous system motor axons over the dorsal and ventral muscle groups stain for NETB protein (Mitchell, 1996 and Harris, 1996).

In the ventral nerve cord of Drosophila most axons are organized in a simple, ladder-like pattern. Two segmental commissures connect the hemisegments along the mediolateral axis and two longitudinal connectives connect individual neuromeres along the anterior-posterior axis. Cells located at the midline of the developing CNS first guide commissural growth cones toward and across the midline. The first growth cones navigate toward the anterior most ventral unpaired median (VUM) cell and thus pioneer the prospective posterior commissure. Only when the posterior commissure is established, the anterior commissure forms. In later stages, midline glial cells, migrating toward the posterior, are required to separate anterior and posterior commissures into distinct axon bundles. The VUM neurons reside ventral to the posterior commissure and project in a characteristic axon-bundle to the anterior commissure. Migration of two midline glial cells occurs along these cell processes. To unravel the genes underlying the formation of axon pattern in the embryonic ventral nerve cord, a saturating ethylmethane sulfonate mutagenesis was conducted, screening for mutations that disrupt this process. Subsequent genetic and phenotypic analyses support a sequential model of axon pattern formation in the embryonic ventral nerve cord. Specification of midline cell lineages is brought about by the action of segment polarity genes. Five genes are necessary for the establishment of the commissures. Two gene functions are required for the initial formation of commissural tracts, in addition to the function of commissureless, the netrin genes, and the netrin receptor encoded by the frazzled gene. Over 20 genes appear to be required for correct development of the midline glial cells which are necessary for the formation of distinct segmental commissures (Hummel, 1999a).

Subsequent analysis has defined four sequential steps involved in commissure development. Initially, single minded, jaywalker, Egf receptor and slit are involved in the first step in midline formation: the formation of the anlage of the CNS midline. Next the segment polarity genes hedgehog, engrailed, patched and wingless are involved in the specification of midline cell number. It is possible that midline and ectodermal pattern formations occur at the same time. In addition to the segment polarity genes other signaling mechanisms appear important. Notch, for example, is required to specify the different midline lineages. The third step in commissure formation consists of the formation of commissures. Once the midline cells have been specified, they guide commissural growth cones toward and across the midline. Here, the Netrins, frazzled, commissureless, weniger, schizo, roundabout and karussel play an essential role. The fourth step in commissure development involves the separation of the commissures. Contrary to midline specification and initial commissure formation, this process occurs relatively late during embryogenesis and thus a maternal contribution is not likely to rescue a mutant phenotype. In addition, the separation of commissures requires not only the differentiation of the midline glial cells but also the differentiation of the midline neurons as well as interactions of these two cell types for normal migration to occur. This might explain the large number of genes identified (Hummel, 1999a).

The analysis of mutations reveals two major phenotypic classes: the pointed and the tramtrack groups. pointed and tramtrack mediate different aspects of glial development. In pointed mutants no glial differentiation occurs, whereas ectopic pointed expression results in ectopic glial differentiation. tramtrack, in contrast, does not interfere with actual glial cell differentiation but appears to be required for the repression of neuronal differentiation in these cells. The pointed group consists of pointed itself, rhomboid, kastchen, klotzchen, kette, schmalspur, mochte gern, spitz, Star, cabrio and kubel. Mutations in eight other genes lead to an axon phenotype initially described for tramtrack. In tramtrack-type mutation (tramtrack, shroud, disembodied, spook, shade, shadow, phantom, and rippchen) commissures appear fused, but in contrast to pointed group mutations, connectives are not affected (Hummell, 1999a).

Most of the neurons of the ventral nerve cord send out long projecting axons that cross the midline. In the Drosophila CNS, cells of the midline give rise to neuronal and glial lineages with different functions during the establishment of the commissural pattern. The development of midline cells is fairly well understood. In the developing ventral neural cord, 7-8 midline progenitor cells per abdominal segment generate about 26 glial and neuronal cells, i.e. 3-4 midline glial cells, 2 MP1 neurons, 6 VUM neurons, 2 UMI neurons, as well as the median neuroblast and its support cells. The VUM neurons comprise motoneurons as well as interneurons, which project through the anterior and posterior commissures. Genetic studies indicate that the VUM neurons are involved in the initial attraction of commissural growth cones. The MP1 neurons are ipsilateral projecting interneurons, which participate in the formation of specific longitudinal axon pathways. The median neuroblast divides during larval and pupal stages. Contrary to what occurs in the grasshopper CNS, the Drosophila median neuroblast does not generate midline glial cells. In Drosophila, the midline glial cells develop from a set of 2-3 progenitors located in the anterior part of each segment. A function of the midline glial cells during the maturation of the segmental commissures has been found, such that two midline glial cells migrate along cell processes of the VUM-midline neurons to separate anterior and posterior axon commissures. If this migration is blocked, a typical fused commissure phenotype develops. Toward the end of embryogenesis, midline glial cells are required for the formation of individual fascicles within the commissures (Hummel, 1999b and references).

Independent of whether Netrin acts by a repulsive or attractive mechanism, evidence is provided that beside the Netrin/Frazzled (DCC) signaling system an additional attractive system(s) is operating in the developing embryonic nervous system of Drosophila. Attractive cues appear to be provided by the midline neurons. The genes schizo and weniger are likely to encode either additional components of the Netrin signaling system or define a second attractive guidance system. In order to obtain further insights in the function of these genes, several double mutant combinations were generated. If schizo or weniger act downstream in the netrin-frazzled pathway, no enhancement of the commissural phenotype would be expected, as compared to the frazzled deficiency phenotype. In embryos homozygous for a hypomorphic frazzled allele or mutant for schizo, only some commissural connections are missing. weniger mutant embryos have a penetrant CNS phenotype and all neuromeres are affected. However, embryos double mutant for frazzled and schizo lack most commissural axons. Similar synergistic effects are seen in frazzled/weniger or in schizo/weniger double mutant embryos. These double mutant analyses also indicate that axons crossing the midline in fra and netrin mutant embryos do not do so because of a loss of a repulsive Netrin signal. In the light of the synergistic effect seen in the frazzled/schizo double mutant it is suggested that, beside Netrin and its receptor, other proteins are required to guide commissural growth cones toward the midline. Furthermore, in the absence of two of the attractive signaling components, the existence is revealed of repulsive functions of the CNS midline. In the double mutant, the repulsive function predominates and directs axons out of the CNS (Hummel, 1999b).

What is the function of midline neurons in commissure formation? Attractive and repulsive signal originating from the midline are required for normal commissure development. The Drosophila midline comprises glial and neuronal cell lineages. These data indicate that these two cell types exert distinct functions during commissure formation. The first commissural growth cones invariably steer toward the anterior-most VUM neurons where these growth cones cross the midline to form the posterior commissure. This indicates that initially the midline neurons attract the commissural growth cones. The netrin genes that encode an attractive signal for commissural growth cones are expressed in midline neurons and glial cells during initial commissure formation. However, the number of commissural fibers is normal in mutations affecting the development of the midline glia. Similarly, ablation experiments using the directed expression of reaper and grim in the midline glial cells result in a fused commissure phenotype and do not lead to a reduction in the number of commissural axons crossing the midline. Thus, it is proposed that the midline glial cells do not play an essential role in attracting the commissural growth cones. The glial derived Netrin signal could be required to counteract repulsive signals. Additional support for the assumption that the midline neurons attract commissural growth cones is provided by the orthodenticle mutant phenotype. Here some midline neurons as well as one of the two segmental commissures is missing. Similarly, expression of dominant negative Jun in all midline cells results in a loss of midline neurons and a concomitant loss of all commissures. Furthermore, in patched mutant embryos the midline glial cells are almost absent and appear to be transformed into midline neurons. Attraction of commissural growth cones is normal in these embryos, however commissural axons stall at the midline. This suggests that the midline glial cells do not participate in attracting commissural growth cones but provide locally acting, contact dependent cues helping growth cones across the midline. Similarly, in the vertebrate neural tube, changes in growth cone morphology have suggested that commissural axons are guided by a contact dependent mechanism across the floor plate (Hummel, 1999b and references).

The following model is proposed for commissure formation. The initial growth of commissural growth cones towards the midline in stage 12 embryos is guided by an attractive signal expressed by the midline neurons. Presumably, this attraction is mediated by early Netrin expression in the midline neurons or alternatively by the action of a Schizo/Weniger attractive system. At this early developmental stage the midline glial cells are elongated in shape, contacting the epidermis with their basal side and are assumed to send out cellular processes contacting the VUM-midline neurons at the dorsal side of the nervous system. The midline glial cells express a repulsive signal that is conveyed to lateral axons via the Robo receptor and/or the karussell gene product. This repulsive function restricts the first axons to cross the midline just anterior of the VUM neurons. The midline glial cells also express a contact dependent permissive guidance cue helping the axons to cross the midline. Subsequently, neuron-glia interaction at the midline results in the migration of the midline glial cells along processes of the VUM neurons (Hummel, 1999b).

Pupal stage

Retinal axons in Drosophila make precise topographic connections with their target cells in the optic lobe. The role of the Netrins and their receptor Frazzled have been investigated in the establishment of retinal projections. The Netrins, although expressed in the target, are not required for retinal projections. Surprisingly, Frazzled, found on both retinal fibers and target cells, is required in the target for attracting retinal fibers, while playing at best a redundant role in the retinal fibers themselves; this finding demonstrates that target attraction is necessary for topographic map formation. Frazzled is not required for the differentiation of cells in the target. These data suggest that Frazzled does not function as a Netrin receptor in attracting retinal fibers to the target; nor does it seem to act as a homotypic cell adhesion molecule. The possibility is favored that Frazzled in the target interacts with a component on the surface of retinal fibers, possibly another Netrin receptor (Gong, 1999).

net A and net B are expressed in identical patterns: both transcripts are expressed in lamina precursors, which in wild type form an arc-shaped ribbon of cells. Thus, the Netrins are expressed in a pattern that would allow them to act as signals for incoming fibers. Fra protein, in contrast, is strongly expressed in photoreceptor axons, suggesting that retinal fibers have the ability to sense Netrin in the target. Interestingly, Fra is also expressed in the target structure, the lamina. fra transcripts are found in an arc-shaped band of cells similar to net transcripts, but double RNA in situ hybridizations reveal that fra and net transcripts do not colocalize to the same cells. Instead, fra transcripts are expressed in more mature lamina precursor cells located posteriorly adjacent to the net-expressing lamina precursor cells. While the transcript is only expressed very transiently, Fra protein expression persists and is thus present throughout the differentiated lamina and in all lamina cells (Gong, 1999).

What is the molecular function of Fra in the target cells? The fact that removal of both Netrins does not affect the retinal projection makes it unlikely that Fra functions as a Netrin receptor in the lamina target. Further, the fact that removal of Fra from the retinal fibers does not affect their projection, makes it unlikely that Fra functions as a homotypic cell adhesion molecule, directly effecting the attractive interaction between retinal fibers and their target cells. Given these findings, a third possibility is favored: Fra in the target cells may interact in a heterotypic fashion with an unidentified component on the surface of retinal fibers. It is possible that this component is another Netrin receptor. This idea is supported by the finding that Netrin misexpression in retinal fibers results in projection defects that phenotypically mimic the removal of Fra from the target, suggesting the presence in retinal fibers of another Netrin receptor in addition to Fra. The existence of additional Netrin receptors in the fly is expected. Apart from an UNC-5 type receptor, which has been found in both worms and vertebrates, a second DCC/UNC-40 homolog may also exist in the fly, based on genetic evidence that UNC40 function is partially redundant in the worm: molecular null alleles of unc40 display a less severe phenotype than some truncation alleles, suggesting that the truncated proteins interfere with a second pathway (Gong, 1999).

Effects of Mutation or Deletion

Deletion of both NetA and NetB gives rise to thinner and sometimes absent commissures and occasional breaks in the longitudinal tracts. The posterior commissure is more severly affected that the anterior commissure. The commissural phenotype is rescued by either NetA or NetB in transgenic flies. Overexpression of NetB throughout the CNS results in thinner commisures than normal, similar to the phenotype observed in deficiency embryos. This suggests that commissural axons are either indifferent to midline Netrin owing to uniform levels throughout the CNS, or are instead now attracted to axons or cell bodies lateral to the midline. Ectopic expression of NetB in all muscles causes the intersegmental nerve to wander, branch, and sometimes stall short of its dorsal targets (Harris, 1996, Mitchell, 1996).

To assess the role of repulsion by Netrins and Unc-5 in shaping motor axon pathways, the development of these trajectories was examined in Df(1)NP5 embryos, in which both the NetA and NetB genes are deleted. For this, the general motor axon marker MAb 1D4 and anti-Unc5 were used. No abnormalities were detected in the SNa and SNc projections in these embryos. The lateral migration of peripheral and exit glia, visualized with anti-Repo antibodies, also appears normal in Netrin-deficient embryos. Double-stranded Unc5 RNA was injected into wild-type embryos in an attempt to specifically disrupt Unc5 function by RNA-mediated interference (RNAi). Although this resulted in a strong reduction in Unc5 staining, MAb 1D4 did not reveal any misrouting of SN motor axons in these embryos. Thus, while the expression data suggest a role for Unc5 in repelling SN motor axons out of the CNS and away from Netrin-expressing muscles, the genetic data indicate that repulsion by Netrins is likely to be just one of multiple guidance forces that control these projections (Keleman, 2001).

However, SNa motor axons can be repelled by Netrins. If NetB is ectopically expressed on all muscles using a 24B-GAL4 driver and a UAS-NetB transgene, SNa axons often stall at the edge of the CNS or fasciculate with the ISN (Mitchell, 1996). Does this gain-of-function phenotype depend on Unc5 function? To test this, Unc5 double-stranded RNA was injected into 24B-GAL4/UAS-NetB embryos. In control embryos that were either uninjected or injected with buffer alone, SNa motor axons fail to enter their lateral muscle target region in 54% or 57% of hemisegments, respectively. In contrast, this phenotype is seen in only 14% of hemisegments in Unc5 RNAi embryos. These data establish that SNa motor axons do indeed sense Netrin as a repulsive signal acting through the Unc5 receptor (Keleman, 2001).

The Drosophila ARF6-GEF Schizo controls commissure formation by regulating Slit: Genetic interaction with the attractive cue Netrin

The CNS of bilateral symmetric organisms is characterized by intensive contralateral axonal connections. Genetic screens in Drosophila have identified only a few genes required for guiding commissural growth cones toward and across the midline. Two evolutionarily conserved signaling molecules, Netrin and Slit, are expressed in the CNS midline cells. Netrin acts primarily as an attractive signaling cue, whereas Slit mediates repulsive functions. A detailed analysis is provided of the Drosophila gene schizo, which is required for commissure formation. schizo leads to a commissural phenotype reminiscent of netrin mutant embryos. Double-mutant analyses indicate that Netrin and Schizo act independently. The schizo mutant phenotype can be suppressed by either expressing netrin in the CNS midline cells or by a reduction of the slit gene dose, indicating that the balance of attractive and repulsive signaling is impaired in schizo mutants. Overexpression of the schizo RNA in the CNS midline using the GAL4/UAS system leads to a slit phenocopy, suggesting that schizo primarily antagonizes Slit signaling. This is further supported by cell type-specific rescue experiments. The schizo gene generates at least two proteins containing a conserved Sec7 and a pleckstrin homology domain (PH) characteristic for guanine nucleotide exchange factors (GEF) acting on ARF GTPases, which are known to regulate endocytosis, In support of the notion that schizo regulates Slit expression via endocytosis, it was found that blocking endocytosis leads to a schizo-like phenotype. It is thus proposed that the balance of the two signaling cues Netrin and Slit can be regulated, controlling membrane dynamics (Önel, 2004).

Only four zygotically active genes were found in a screen for mutations affecting commissure formation (frazzled, weniger, schizo and the netrin gene complex). Two EMS-induced schizo mutants (schizo^C1-28 and schizo^U112) were initially identified. Subsequently two P-element induced schizo alleles (schizo^l(3)3 and schizo^P244) were identified. All these alleles led to a reduction in the number of commissural fibers crossing the CNS midline. Interestingly, the anterior commissures were affected more prominently. Not all neuromeres were equally affected and the strongest defects were generally observed in abdominal segments A1-A4. All CNS midline cells formed in normal number in the absence of schizo function. However, as generally observed in mutants affecting formation of commissures, the midline glial cells migrated out laterally along the few remaining commissural fibers. In addition to the commissural phenotype, defects in the longitudinal connectives were noted (Önel, 2004).

The most prominent function of schizo is its role in commissure development. Two major signaling cascades are known to control axonal growth across the midline. They are initiated by the signaling molecules Netrin and Slit, which are both secreted by the CNS midline glial cells in the Drosophila embryo. First genetic interaction studies of schizo and frazzled or schizo and netrin function demonstrate a much stronger commissural phenotype in double mutants than embryos mutant only for schizo, frazzled or netrin. The commissural phenotypes of the double-mutant embryos suggest that schizo is not acting within the Netrin signaling pathway but may be required for a Netrin-independent attractive pathway. Alternatively, schizo may be necessary for suppressing the perception or the generation of a repulsive signal normally generated by the CNS midline cells (Önel, 2004).

The main axonal repulsive signal is encoded by slit. Slit is an LLR protein secreted by the CNS midline glial cells. schizo function appears to be required to downregulate repulsive signaling, either by affecting the generation of active Slit protein or by preventing signaling in the commissural growth cones; the mutant schizo phenotype could be explained by an upregulated Slit signaling. Thus, one might expect that the schizo commissural phenotype could be suppressed by a concomitant reduction in the dose of slit function. slit^-/+; schizo^–/– as well as robo^-/+; schizo^–/– embryos were generated, and in both cases a suppression of the schizo CNS phenotype was observed. Thus schizo might be required to negatively regulate Slit signaling (Önel, 2004).

If schizo is indeed a negative regulator of slit function, an increase of schizo gene dose should result in a decrease of active Slit signaling. One might thus be able to enhance the mutant slit phenotype by using a schizo gene duplication. Following mapping of schizo to the base of the left arm of chromosome 3 a chromosomal translocation of the corresponding part of the third chromosome to the Y chromosome (Tp(3;Y)A81) was used to generate embryos with three copies of schizo. In an otherwise wild-type background, this triplication of the schizo region did not result in an abnormal CNS phenotype. However, when the schizo translocation was placed in a heterozygous slit^–/+ background, a slit-like phenotype was observed that was never detected in heterozygous slit embryos (Önel, 2004).

schizo maps to chromosome region 78A/B between the genes poils aux pattes and knockout. To identify the schizo gene in this chromosomal interval P-element-induced schizo alleles were used. The chromosomal insertions of the P-elements in l(3)3 and P224 were determined by inverse PCR and Southern analyses and the results suggested that schizo corresponds to CG32434. The lethality associated with the P-element-induced l(3)3 schizo mutation could be reverted by precise excision of the P-element. Mutant l(3)3 embryos displayed a schizo phenotype with reduced commissures and defective fasciculation in the longitudinal connectives. Subsequent sequencing of cDNA clones LP01489, RE44556 and GH10594 isolated by the BDGP showed that the schizo locus encompasses 41 kb of genomic DNA. At least two different promoters direct the expression of two isoforms of 1325 amino acids (SchizoP1) and 1313 amino acids (SchizoP2) in length. Verification of the cloning of schizo was obtained by genetic rescue experiments. These deduced schizo proteins correspond to the Iso1 and Iso2 variants of the loner gene, which was recently identified in a screen for mutations affecting mesoderm development. By contrast Chen (2003) GH10594 was found to be entirely contained within the LP01489 sequence and no evidence was found for a third schizo protein isoform (Önel, 2004).

The deduced Schizo proteins share three conserved sequence modules. In the N-terminal region there is a so-called IQ domain, which is predicted to interact with calmodulin. Within the C-terminal third of the protein a Sec7 domain is directly adjacent to a PH-domain. Proteins characterized by such a domain signature are generally acting as guanine nucleotide exchange factors (GEFs). The Anopheles homolog is about 90% identical. The closest human homologs are EFA6, being 32% identical to Schizo, lacking the IQ domain, and ARF-GEP₁₀₀ showing a 40% identity to Schizo. Both human proteins were shown to act as ADP ribosylation factor 6 (ARF6)-GEFs suggesting that schizo might have a similar function (Önel, 2004).

The molecular identification of schizo allowed for the determination of the expression pattern throughout development. schizo expression is already detected in the unfertilized egg, indicating a prominent maternal contribution. schizo expression stays almost uniform until the end of stage 10. Within the developing nervous system, expression can be noted in the CNS midline cells. In addition, schizo expression can be detected in the epidermis and the visceral mesoderm (Önel, 2004).

The commissural schizo phenotype does not allow the deduction of cell type in which Schizo normally acts. To test the cell-type requirement the GAL4 system was used and UAS-schizoP1 and UAS-schizoP2 transgenic flies were established. Expression of the different schizo proteins was directed in the CNS midline cells of mutant schizo embryos using the sim-GAL4 or sli-GAL4 driver strains. In both cases expression could rescue the schizo mutant CNS phenotype indicating that Schizo acts in the midline glial cells, which express both Slit and Netrin (Önel, 2004).

Genetic data indicate that schizo impairs Slit signaling in the CNS. This was further supported by overexpression of schizo. Whereas expression of schizo (P1 or P2) in all CNS midline cells of wild-type embryos did not evoke an abnormal phenotype, the same expression of schizo in heterozygous slit mutant embryos was able to induce a mild slit phenocopy. These results were similar to the ones obtained using chromosomal translocations, supporting the notion that Schizo acts in the CNS midline by regulating the level of Slit expression (Önel, 2004).

To reduce Slit activity, schizo might suppress exocytosis of Slit-containing vesicles, or it might promote endocytosis of Slit-containing vesicles from the membrane. Work from vertebrate tissue culture models has shown that Arf-GEFs such as Schizo can activate endocytosis. To test whether endocytosis might be relevant for commissure formation a dominant negative Shibire protein was expressed; this efficiently blocks endocytosis specifically in the CNS midline cells using the sim-GAL driver. In about 60% of such embryos a schizo phenocopy was observed. When higher levels of the dominant negative Shibire protein were expressed using the rho-GAL driver all embryos developed a schizo phenocopy, suggesting that endocytosis participates in the regulation of Slit function in the CNS midline cells. To further support the notion that Schizo induces endocytosis of Slit, the negative Shibire protein was expressed in a heterozygous slit mutant background. This indeed led to clear suppression of the Shibire-induced phenotype. Schizo and its vertebrate homologs exert at least part of the function through the small GTPase Arf6. Arf6 mRNA is supplied maternally and is expressed ubiquitously during embryonic development. To determine whether Schizo acts via Arf6 to control endocytosis of Slit by the midline glial cells, a dominant negative Arf6 construct (Chen, 2003) was expressed. Following expression in the midline cells using the sim-GAL driver no mutant phenotype was observed. Following expression of higher levels of Arf6^DN using the rho-GAL driver, about 15% of the embryos developed a schizo phenocopy, supporting the notion that Schizo acts via Arf6-regulated endocytosis to control the level of Slit expression on the midline glial cells (Önel, 2004).

Thus genetic and molecular data support a model in which schizo negatively regulates the expression of Slit in the CNS midline cells. This study shows that schizo acts in a rather similar way to commissureless; however, rather than affecting the Roundabout receptor, schizo appears to act on the expression of the Slit ligand. First it was found that the triplication of the schizo gene interfers with slit function and that reduction of slit expression in schizo mutant embryos rescues the schizo mutant phenotype. Finally, expression of a schizo transgene in the Slit-expressing CNS midline cells (1) was able to rescue the schizo mutant phenotype and (2) could induce a slit phenocopy when expressed in wild-type embryos. The deduced nature of the Schizo protein suggests that it affects Slit expression by post-transcriptional mechanisms (Önel, 2004).

Guanine-nucleotide exchange factors (GEFs) help to convert the inactive GDP-bound form of small GTPases into a GTP-bound active form. Schizo is a new Sec7 domain containing GEF, which shows 40% homology to human Arf-GEP₁₀₀. Arf-GEP₁₀₀ localizes to endosomal membranes and promotes GDP/GTP exchange on ARF6. The small GTPase ARF6 is a plasma membrane-localized protein and functions in the regulation of membrane ruffling, cell motility, aspects of endocytosis and exocytosis, membrane recycling, reorganization of the cortical actin cytoskeleton and activation of phospholipase D. In Drosophila, Arf6 is remarkably well conserved, being more than 96% identical to the human counterpart (Önel, 2004).

One aspect that might hint at how Schizo regulates Slit expression is the role of ARF6 in endocytosis and exocytosis. The function of ARF6 in endocytosis is twofold. It either regulates clathrin-mediated endocytosis at the apical surface of polarized epithelial cells or it is able to regulate non-clathrin-mediated endocytosis and the recycling pathway in non-polarized cells. ARF6 has also been postulated to play a role in Ca²⁺-activated dense core vesicle (DCV) exocytosis by regulating phosphatidylinositol(4,5) biphosphate (PIP₂). Overexpression of a UAS-ARF6 construct in midline glia cells does not result in a schizo-like phenotype, whereas expression of a dominant negative form of Arf6 results in a phenocopy of several phenotypes associated with the schizo mutant (Chen, 2003). This suggests that Arf6 might also be involved in the regulation of Slit expression (Önel, 2004).

In-vivo Slit and Netrin are both expressed by the same CNS midline cells and their expression needs to be in an intricate balance. The importance of this balance and not the individual expression levels is highlighted by the fact that it is possible to rescue the schizo mutant phenotype by both increased Netrin expression or reduced Slit expression. Within the midline glia, however, Schizo appears to primarily affect Slit expression either by inducing its endocytosis and subsequent degradation or by blocking exocytosis and thus release of Slit (Önel, 2004).

The latter case would suggest that Slit and Netrin are brought to the membrane of the midline glial cells in distinct vesicle populations, whereas the former case would require a specific membrane receptor for the Slit protein expressed by the CNS midline glia. Given the fact that the secreted Slit protein is found at very high levels at the midline glial cell membrane, this appears probable. Moreover, expression of a dominant negative Shibire protein in the midline glia leads to a schizo phenocopy. shibire encodes the Drosophila dynamin and is required for endocytosis and a block of shibire function leads to a block of endocytosis, which might result in higher levels of Slit expression. Thus, regulation of membrane dynamics appears crucial in controlling the function of the signaling molecule Slit (Önel, 2004).

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date revised: 25 May 2008

 

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