GlcAT-P in Nervous System Development Figure 6. brv mutant larvae display shorter peripheral nerves. Representative pictures of L1, L2 and L3 larval brains from brv+ (A, B, C) and brv1 mutants (D, E, F) expressing Nrv2-GAL4;UAS-GFP(S65T) to visualize the peripheral nerves. White arrowheads indicate the most posterior projecting and longest peripheral nerves used for these measurements. Histograms (G, H, I) compare the average lengths of the longest peripheral nerves during L1 (G), L2 (H) and L3 (I) larva between wild-type and brv1 mutants. The peripheral nerves are significantly shorter in brv1 mutants (*: p,0.0001). (J–S) Representative frames, taken from time-lapse recordings of wandering larva expressing Nrv2-GAL4; UAS-GFP(S65T), display the VNC during one full cycle of a larval contraction and expansion during locomotion. A brv+ (J–N) and brv1 larva (O–S) is shown. Ripples (arrowheads in P, Q), indicative for relaxation from tension, were detected only in VNC of brv1 mutants but not in brv+ (J–N). Bar: 100 mm. A time line (in sec) is shown below panels J–S. doi:10.1371/journal.pone.0028106.g006 motoneurons) and glial cells in the larval CNS. The brv mutation neither impaired the viability of these neurons nor resulted in obvious developmental defects, apart from the longitudinal displacement of their cell bodies and neurites due to VNC elongation. During Drosophila nervous system development, the VNC undergoes a process of condensation at late embryonic stages [54]. Recently, a mutation in the C1GalTA gene, which codes for a core 1 galactosyltransferase, has been isolated and its phenotype characterized as a defect in embryonic VNC condensation [55]. C1GalTA is one of the several C1GalTs genes involved in mucin- type O-glycosylation in Drosophila [55]. Interestingly, the C1GalTA mutant phenotype is almost indistinguishable from brv mutants with regard to the larval VNC extension. However, we have not detected embryonic VNC condensation defects in brv mutants. We attribute this difference to the expression of C1GalTA, but not GlcAT-P, in the embryonic CNS [55]. GlcAT-P controls the length of the VNC and the peripheral nerves We observed a reverse correlation between the length of peripheral nerves and the extension of the VNC in brv mutants. It has been reported that tension on nerves during growth might stimulate the elongation of axons [5]. It is conceivable that if tension on nerves generated by the growing larval body cannot be counteracted by concomitant growth of the peripheral nerves, the VNC would be forced to compensate such tension by stretching, thus resulting in the VNC extension phenotype observed in brv mutants. Such notion is supported by the retraction of the VNC in brv mutant larvae after severing the very posterior segments. This procedure liberates the attachment of the most posterior peripheral nerves to the posterior segments of the larva and at least partially releases the tension on the nerves and the VNC. Thus, our data is consistent with the hypothesis that GlcAT-P plays a role in peripheral nerve growth during Drosophila larval development. Alternatively, GlcAT-P might be required in the VNC during larval stages to keep the VNC compacted. However, our finding that the VNC extension is significantly reduced in brv-/-, Tb+/- mutants as compared to brv-/- mutants indicates that the VNC is not undergoing an active extension in brv mutants and that the elongation is clearly dependent on the total length of the larval body. This observation suggests that GlcAT-P is not required for VNC compaction since the VNC should have been elongated to a similar extent in the Tb+/- background if GlcAT-P would function in VNC compaction. Nevertheless, it cannot be completely ruled out that brv mutation causes enhanced VNC elasticity due to changes in the ECM deposited into the VNC by hemocytes and maybe glia. As a consequence of reduced VNC rigidity and its extension, the peripheral nerves do not bear significant strain and hence are not induced to grow. It is also feasible that both a defect in peripheral nerve growth as well as in the elasticity of the VNC contribute to the extension of the VNC. However, even in the context of the alternative explanation it is still obvious that the final consequence is that peripheral nerves in brv mutants do not elongate as much as they would in the wild type (wt). During larval growth the terminals of axons in the peripheral nerves progressively move away from the nervous system predominantly as a consequence of the extension of the larva along the anterior-posterior body axis. During wt larvae growth the length of the VNC does not elongate much whereas the peripheral nerves increase quite dramatically in length. This reveals a mechanism which promotes extension of integrated peripheral axons during larval growth. In contrast, in brv mutants a significant reduction of the length of peripheral nerves is observed and we suggest that due to the reduced growth of peripheral nerves the VNC compensates the resulting tension by elongating. PLoS ONE | www.plosone.org 9 November 2011 | Volume 6 | Issue 11 | e28106