GlcAT-P in Nervous System Development Figure 8. repo interacts genetically with GlcAT-P. Representative pictures from brv+ (A), brv1 (B), repo-/+, brv1-/- L3 (C) and repo-/-, brv1-/- L2 larvae. The CNS is visualized either by Nrv2-GAL4;UAS-GFP(S65T) (A, B) or by repo-GAL4;UAS-GFP(S65T) (C–D). White arrowheads indicate the tip of the VNC. (E, F) Histograms compare the average lengths of VNCs (E) and the most posterior peripheral nerves (F) for L3 as well as L2 larvae. repo-/+, brv1-/- VNCs are significantly longer as compared to brv1-/- mutants (*: p,0.0001). Bar: 100 mm. doi:10.1371/journal.pone.0028106.g008 addition, some proteoglycans and glycolipids are also known to bear the HNK-1 epitope [20], and in mice, the HNK-1 epitope is selectively found on myelinating Schwann cells associated with motor axons [19]. In GlcAT-P/b3gat1 knock-out mice, an almost complete loss of the HNK-1 epitope in the brain is observed [35]. In our analysis the HNK-1 epitope was still detected in immunoblots of brv mutant larval CNS, suggesting that in the fly, GlcAT-P is not required or is redundant for the generation of the HNK-1 epitope. Indeed, GlcAT-S has been shown to be involved in HNK-1 epitope synthesis as well [24] and we have demonstrated that GlcAT-S protein is present in the fly larval- CNS. Therefore, in Drosophila at least a partial redundancy between GlcAT-P and GlcAT-S might explain the overall presence of the HNK-1 epitope. It is worth noting that despite the apparent lack of changes in global levels of the HNK-1 epitope, the VNC extension phenotype was still completely penetrant in brv mutant larvae. Hence, the extended VNC phenotype in brv mutant larvae could be independent of the HNK-1 epitope. This is also supported by the similarity to the phenotype of C1GalTA which might indicate that GlcAT-P, like C1GalTA, is also involved in generation of glucuronylated core 1 O-glycans [67,68]. Alterna- tively, our analysis of HNK-1 expression levels might have overlooked a specific HNK-1 carrying protein responsible for the VNC extension phenotype, and as such VNC extension could still be a result of a specific, yet unknown, protein lacking the HNK-1 epitope as a consequence of the loss of GlcAT-P activity. All three brv alleles identified in the EMS screen affected the C-terminal catalytic domain of GlcAT-P and ectopic expression the GlcAT- Pcd was not able to rescue brv mutants. We conclude that loss of catalytic GlcAT-P activity is sufficient to result in a functional mutation and the catalytic activity of GlcAT-P is crucial for peripheral nerve growth. Interestingly, one of the currently identified GlcAT-P protein targets in vertebrates is the HNK-1 epitope carrying ECM glycoprotein tenascin-C [67]. Ten-C is found in glial cells and potentially promotes or inhibits neurite outgrowth in a context dependant manner [69,70]. Glial cells and neurons/axons are also known to interact with each other [71] by glia supporting axonal functions e.g., by neurotrophic and metabolic support [72] as well as by providing the extracellular matrix context [69,70]. Based on these reports and our own observations, we suggest a model in which GlcAT-P is critical in hemocytes possibly to deposit specific ECM on peripheral nerves. The ECM then in turn is critical to peripheral glia for stretch growth of the peripheral nerves for example by directly or indirectly being involved in the incorpo- ration of new membrane material to growing axons thereby facilitating stretch growth of integrated axons during larval growth. It has been shown that ECM is more than a mere structural support to cells and instead is involved in signaling on the molecular level via ECM receptors such as integrins, dystroglycans, membrane associated heparan sulphate proteogly- cans (HSPGs), glycipicans and syndecans [73,74,75]. For example, integrins are crucial components of cell adhesion and together with other ECM receptors can signal to the cytoskeleton of cells leading to cell shape changes, regulation of cell proliferation, cell migration, apoptosis and differentiation. Furthermore, a clear link between ECM and axonal growth and growth cone pathfinding has been established [73]. In this context the ECM has been shown to regulate actomyosin contractility, actin polymerization and actin stabilization and therefore might impact on the cytoskeleton of signaling receiving cells via Src, FAK, Rac1, RhoA and Rock leading to modified growth cone pathfinding [76,77]. Therefore, it is possible that in the context of the extended ventral nerve cord and the shorter peripheral nerves in GlcAT-P PLoS ONE | www.plosone.org 11 November 2011 | Volume 6 | Issue 11 | e28106