Figure 7. In brv mutant larvae the VNC appears to be under tension of peripheral nerves. Representative pictures of L2 larvae from brv+ (A, B) and brv1 mutants (C, D) expressing Elav-mCD8-GFP before (A, C) and after (B, D) dislodging the connections of the posterior most peripheral nerves. Arrowhead in (D) points towards a kink that appeared during relaxation of the VNC in brv1 mutants. Representative pictures from brv+ (E), brv1-/- (F), Tb1-/+ (G) and brv1-/-, Tb1-/+ mutant (H) L3 larvae expressing Nrv2-GAL4;UAS-GFP(S65T). The e\tension of the VNC in brv1-/-, Tb1-/+ is reduced as compared to brv1-/-. Histograms (I, J) quantify the average lengths of VNCs and posterior-most peripheral nerves. brv1-/-, Tb1-/+ VNCs are significantly shorter as compared to brv1-/- mutants (*: p = 0.0005). White arrowheads indicate the posterior tip of the VNC. Bars: 100 mm. doi:10.1371/journal.pone.0028106.g007 Taken together, we conclude that GlcAT-P controls the elongation of peripheral nerves/axons either indirectly via regulating the compaction of the VNC or by directly regulating the growth of peripheral nerves and the axons therein. It is worthwhile to note that most organisms with a nervous system exhibit elongation of the anterior posterior body axis during development which normally goes beyond the growth of the nervous system along the same axis. The contacts between e.g. centrally located motoneurons and their peripheral muscles are generally established before such extensive growth phases. A mechanism of ‘‘stretch growth of integrated axon tracts’’ [5] has been postulated to allow peripheral nerves and the integrated axons in these nerves to grow in order to compensate for possible tension arising from the growth process of the organism on the central nervous system. Although such stretch growth of integrated axon tracts seems to be fundamental to all growing organisms, almost nothing is known about the cellular and molecular mechanisms driving this process. Our data suggests that GlcAT- GlcAT-P in Nervous System Development P is involved in this mechanism as in the absence of GlcAT-P function peripheral nerve growth is severely retarded. GlcAT-P may regulate growth of integrated nerves via glia and hemocytes We also have demonstrated that GlcAT-P function is important in hemocytes since targeted expression of GlcAT-P in hemocytes fully rescued the VNC extension as well as pupal lethality whereas neither expression in neurons nor in glia could rescue. Hemocytes are known as versatile cells involved in immune response as well as in immune system unrelated developmental mechanisms [32]. Interestingly, hemocytes have been shown to be required for brain morphogenesis particularly in the process of embryonic VNC condensation [54]. The onset of VNC condensation coincides with the deposition of Collagen IV by hemocytes and a lack of hemocyte migration is associated with a severe reduction in ECM components [54] demonstrating that one major function of hemocytes is ECM deposition. This is further supported by findings that hemocytes express several ECM components such as Peroxidisan, dSPARC, and the proteoglycan MDP; also it has been shown that hemocytes produce structural components of the basement membrane such as laminin A and collagen IV [56,57]. GlcAT-P could have a role in modifying ECM components produced in hemocytes by adding sugar residues to such proteins which in turn might influence cells directly exposed to such modified ECM. We have also shown that partial or complete loss of repo function in brv mutant background results in further and progressive extension of the VNC depending on the repo gene dose. These observations clearly reveal that although GlcAT-P function is required in hemocytes, the phenotypic manifestation of VNC extension in brv mutants also involves functional glial cells. repo affects glia differentiation as well as maintenance of glial function [58,59,60]. Our finding indicates that if the population of glial cells is genetically weakened by reducing or removing Repo function in a brv-/- mutant background, the VNC extension phenotype is enhanced. Strikingly, in specimen double mutant for brv and repo, the extended VNC stretches along the entire body axis of the larva. This is comparable to the VNC extension in late embryos suggesting that in brv-/-, repo-/- double mutants, the growth of peripheral nerves is almost completely abolished thus leading to the extreme extension of the VNC during larval growth. Furthermore, the genetic interaction between brv and repo establishes an important role for glia in stretch growth of peripheral nerves and/or VNC elasticity. Interestingly, the peripheral nerves are covered by a thick layer of ECM [61]. As such, the ECM might be an important provider of contextual information for peripheral glia as well as neurites projecting through the nerves. Some peripheral glial cells also express GlcAT-P which might suggest that glial cells are also involved in either generating their own matrix environment or directly control neurite extension. However, expression of GlcAT-P specifically in glial cells did not rescue the brv mutant phenotype and as such GlcAT-P function in glia is not critical for peripheral nerve growth. Thus, we hypothesize that GlcAT-P has a role in regulating the elongation of integrated nerves and peripheral axons during development via hemocytes and glia possibly by glycosylating and depositing ECM related proteins. GlcAT-P which is mainly expressed in the brain in vertebrates [30] has been shown to be involved in the biosynthesis of the HNK-1 epitope [26] on integral membrane glycoproteins such as the neural cell adhesion molecule [62], myelin-associated glycoprotein [63], L1 [62], transiently expressed axonal glycoprotein-1 [64], P0 [65] and Ependymin [66]. In PLoS ONE | www.plosone.org 10 November 2011 | Volume 6 | Issue 11 | e28106