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First published online 4 March 2009
doi: 10.1242/dev.032847

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Drosophila Neurexin IV stabilizes neuron-glia interactions at the CNS midline by binding to Wrapper

Tobias Stork^*, Silke Thomas, Floriano Rodrigues, Marion Silies, Elke Naffin, Stephanie Wenderdel and Christian Klämbt

Institut für Neurobiologie, Universität Münster, Badestrasse 9, D-48149 Münster, Germany

View larger version (64K): [in this window] [in a new window]   Fig. 1. Localization of Neurexin IV during the formation of commissures. Frontal, anterior upwards (A,C,E,G), and sagittal, anterior leftwards (B,D,F), views of developing commissures in the Drosophila embryo. To detect Neurexin IV localization, we used the GFP-exon trap insertion 454 and subsequent GFP staining (green in A'',B'') or used specific Neurexin IV antibodies (green in C'',D'',E'',F'',G'',G'''). The shape of the midline glial cells (mg) is highlighted by staining for Wrapper expression (red). Neuronal membranes are stained by anti-HRP antibodies (blue). Single confocal sections are shown. B''',D''',F''' schematic representations of B'', D'' and F''. The midline glia (mg) are shown in red, midline neurons are depicted in blue and green highlights Neurexin IV expression. (A,B) In stage 12 embryos, the midline glial cells are still anterior to where the commissures cross the midline. Prominent Neurexin IV expression can be detected in neurons and their axonal projections. (C,D) In stage 14 embryos, both segmental commissures (ac, anterior commissure; pc, posterior commissure) have been established. The midline glial cells are migrating between the commissures. Neurexin IV expression accumulates at neuron-glia contact sites at the CNS midline (arrowheads). The outlined area in C'' is enlarged in G-G'''. (E,F) In stage 16 embryos, the midline glial cells have finished the wrapping of the segmental commissures and have extended processes within the commissural tracts. An intense overlap of Neurexin IV and Wrapper at axon-glia contact sites can be observed (arrowhead). (G) Neurexin IV accumulates at the interface of neuronal cell bodies (asterisks) stained with HRP (G', blue in G'' and G''') and the midline glia are highlighted by Wrapper staining in red (G''').

View larger version (133K): [in this window] [in a new window]   Fig. 2. Neurexin IV is required for axonal wrapping. Frontal views of CNS preparations of stage 16 embryos, the genotype is indicated, anterior is upwards. Axonal membranes are shown in blue (HRP staining), the shape of the midline glial cells is marked by Wrapper expression (red). Single confocal sections are shown. (A',B') Orthogonal sections through the z-stack of confocal images. The plane of section is indicated by a broken line in A,B. (A) In the wild-type CNS, anterior (ac) and posterior (pc) commissures are clearly established and axonal membranes are wrapped by Wrapper-positive midline glial cell processes. (A') In the orthogonal view of the midline the intense ingrowth of midline glial cell processes into the commissural tracts can be seen. (B) In Neurexin IV mutant embryos, commissures form normally, but the midline glial cells fail to establish processes within the commissural axon tracts (arrowhead). (B') In the orthogonal section, it becomes obvious that the midline glial cells grow around the commissural axon bundle but do not establish wraps of individual axonal fascicles (arrowhead). (C-E) In mutant neuroglian (C), coracle (D) or lachesin (E) embryos, neither the formation of commissures nor the wrapping of the commissural axons is affected. The midline glial cells establish intensive contacts with commissural axons.

View larger version (132K): [in this window] [in a new window]   Fig. 3. Neuronal expression of Neurexin IV rescues the Neurexin IV midline glial phenotype. Frontal views of CNS preparations of stage 16 embryos, anterior is upwards, the genotypes are indicated. Axonal membranes are shown in blue (HRP staining), the shape of the midline glial cells is marked by Wrapper expression (red). Neurexin IV expression is shown in green. Single confocal sections are presented. (A) The phenotype of Neurexin IV mutants is not rescued by re-expressing Neurexin IV in all midline cells using the EP-insertion EP604 and simGal4 activation. (B) Re-expressing Neurexin IV in all neurons using the EP-insertion EP604 and elavGal4 activation gives full phenotypic rescue. Note that, sometimes, glial processes extend slightly further into the commissures, as observed in wild type (arrowheads in A,B; Fig. 2A). (C) A partial rescue of the Neurexin IV mutant phenotype is observed by re-expressing Neurexin IV in the eagle pattern (the pattern of expression is shown in the inset in the top right-hand corner). Arrowhead indicates Wrapper-positive midline glial cell processes extending along the Neurexin IV-positive commissural fascicles. (D-D'') After re-expression of Neurexin IV on few ipsilateral-projecting neurons using the connectinGal4 driver, we frequently noted extension of midline glial cell processes into the longitudinal connectives (arrowheads). (E-E'') Likewise, we noted extensions of midline glial cells along commissural axons following eagleGal4-mediated activation of Neurexin IV expression.

View larger version (96K): [in this window] [in a new window]   Fig. 4. Glial Wrapper expression stabilizes neuronal Neurexin IV. (A-C'') Frontal views of CNS preparations of stage 16 embryos, anterior is upwards, the genotypes are indicated. (D-D'') Hindgut epithelium. Axonal membranes are shown in blue in the merge (HRP staining), the shape of the midline glial cells is marked by Wrapper expression (red). Neurexin IV expression is shown in green. Single confocal sections are presented. (A-A'') In wild-type embryos, an intensive overlap of Neurexin IV and Wrapper expression at the CNS midline can be seen. Note the presence of Neurexin IV expression in the commissural axon tracks (arrowhead). The asterisk in A indicates weak Neurexin IV staining on neuronal cell bodies. (B-B'') In Df(2R)02311 mutant embryos that lack wrapper expression, Neurexin IV expression is lost at the midline. By contrast, Neurexin IV expression in CNS neurons can still be detected. Note the weak Neurexin IV expression in the neuropil (np). (C-C'') Following overexpression of Wrapper in all CNS neurons using elavGal4, Neurexin IV is depleted from the CNS midline despite the presence of normal levels of Wrapper expression at the midline glial cells. No Neurexin IV expression can be detected in commissural axons at the midline (arrowheads). Instead Neurexin IV expression is redirected to the neuronal cell bodies (asterisk). (D-D'') Hindgut of a wild-type embryo ectopically expressing Wrapper protein in the ventral cells using the enGal4 driver. Wrapper is recruited to the septate junctions that normally express Neurexin IV.

View larger version (29K): [in this window] [in a new window]   Fig. 5. Neurexin IV encodes two differentially expressed protein isoforms. (A) Schematic representation of the Neurexin IV locus. Transcription is from left to right. The position of the EP604 insertion and the differentially spliced exons 3 and 4 is indicated. Exon 3 has a unique XbaI site, exon 4 carries a unique NcoI site. The arrows indicate the position of the primers used in the RT-PCR experiments shown in B,C. Scale bar: 1 kb. (B) Following expression of a human Flag-tagged PABP protein in either glial cells (repo>hPF) or neuronal cells (elav>hPF), tissue-specific mRNA was isolated. The expression of several genes was assayed using RT-PCR. As expected, repo mRNA is enriched in the repo>hPF RNA pool, whereas elav mRNA is enriched in the elav>hPF pool. crumbs mRNA is specifically expressed in epidermal cells and was used as a negative control and only some crumbs mRNA could be detected in the repo>hPF RNA pool. Neurexin IV mRNA was identified in both the glial and the neuronal mRNAs. (C) To discriminate which of the two Neurexin IV mRNA isoforms was amplified in the two RNA pools, we digested the DNA with NcoI or XbaI, or both. This analysis demonstrates that in the glial mRNA pool, the Neurexin IV exon 3 isoform predominates, whereas in the neurons, the exon 4 predominates.

View larger version (91K): [in this window] [in a new window]   Fig. 6. Wrapper preferentially interacts with Neurexin IVexon4. Drosophila S2 cells expressing the different proteins as indicated were tested for aggregation. (E-L) Red, Wrapper expression; blue, Neurexin IV expression detected with an antibody recognizing both isoforms; green, Myc-tag expression. (A) Upon mixing of Nrxexon3- and Nrxexon4-expressing cells, no aggregation of S2 cells can be detected. (B) Likewise, no aggregation was observed upon expression of Wrapper. (C) When Nrxexon3- and Wrapper-expressing cells were added, aggregates form. (D) Similar aggregates formed when Nrxexon4- and Wrapper-expressing cells are mixed. (E,F) To identify the different cells, we used antibodies against Wrapper (red) and Neurexin IV (blue). (E) When Neurexin IVexon3- and Wrapper-expressing cells, or (F) Neurexin IVexon4- and Wrapper-expressing cells were mixed, Neurexin IV- and Wrapper-expressing cells alternate in the cell aggregates. (G-I) Upon mixing of Wrapper-expressing cells with cells that either express Nrxexon3 or Nrxexon4-myc, aggregates were noted that almost exclusively contained Wrapper- and Nrxexon4-myc-expressing cells. (J-L) When Wrapper-expressing cells were mixed with cells that either express Nrxexon3-myc or Nrxexon4, aggregates form. Again, the aggregates comprise almost exclusively Wrapper- and Nrxexon4-expressing cells. In J, a single Nrxexon3-myc-expressing cell can be seen attached to a cluster of Wrapper and Nrxexon4 cells (arrowhead).

View larger version (103K): [in this window] [in a new window]   Fig. 7. Differential rescue abilities of the Neurexin IV isoforms. Frontal views, anterior is upwards, projection of confocal stacks of stage 16 ventral nerve cords spanning the commissural width stained for the presence of Wrapper (red) to label the CNS midline cells and HRP (blue) to label axonal trajectories. (A) In wild-type embryos, the midline glia engulfs the entire commissure and sends processes across both anterior and posterior commissures (ac, pc; arrowhead). (B) In mutant Neurexin IV embryos, the midline glial cells fail to wrap the segmental commissures (48 out of 50 neuromeres). No midline processes are associated with the posterior commissures (pc, arrow). In addition, only few glial processes are found around the anterior commissure (asterisk). (C) In Neurexin IV mutant embryos expressing the Nrx-IVexon3 protein from the 51D landing site in the elav pattern, the commissural wrapping is only partially rescued and in 48 out of 60 neuromeres, the midline glia fails to cover the posterior commissures (arrow). Anterior commissures often contain only few glial processes (asterisk). Arrowhead indicates commissure partially covered by midline glial processes. (D) In Neurexin IV mutant embryos expressing the Nrx-IVexon4 protein from the 51D landing site in the elav pattern, the commissural wrapping is almost entirely rescued and, in 50 out of 52 neuromeres, the midline glia covers the posterior commissures (arrowhead).

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