essential process (reviewed in [19]). Glycosyltransferases, which transfer monosaccharide units to an acceptor molecule, are responsible for the synthesis of carbohydrate moieties on proteins. The carbohydrate epitope Human Natural Killer 1 (HNK-1), found on several cell adhesion molecules such as the neural cell adhesion molecule (NCAM; [20]), is spatially and temporally regulated during development of the central and peripheral nervous systems. HNK-1 is assumed to be involved in cell-cell interactions such as cell adhesion [21], migration [22] and neurite extension [23]. The HNK-1 epitope, comprising of a unique trisaccharide structure, HSO3-3GlcAb1-3Galb1-4GlcNAc-, is sequentially synthesized by the glucuronosyltransferase P (GlcAT-P) or the glucuronosyltransferase S (GlcAT-S) and by a specific sulphotransferase (HNK-1ST) [24,25,26,27,28]. The HNK-1 carbohydrate is also crucial for maintaining proper neural function [29]. GlcAT-P glucuronyltransferase activity is also required for proteoglycan and glycoprotein biosynthesis [30,31]. Another class of proteins modified by GlcAT-P is extracellular matrix (ECM) proteins and some ECM proteins are known to bear the HNK-1 epitope [20]. In Drosophila, production and secretion of several ECM molecules is an essential function of hemocytes [32]. Besides ECM deposition, hemocytes are also involved in the clearance of apoptotic cells. Possibly due to the above mentioned functions, hemocyte migration along their normal routes and arrival at their correct target tissues is vital for development and survival [32]. A single GlcAT-P ortholog is present in metazoan genomes, including humans, although, three paralogs have been characterized in almost all metazoans: GlcAT-P (also termed b-1, 3-glucuronyltransferase 1 (B3GAT1)), GlcAT-S and GlcAT-I [33]. GlcAT-P has been described as the predominant glycosyltransfer- ase for HNK-1 epitope biosynthesis in the brain. Human GlcAT-P (B3GAT1) has been implicated as a candidate gene for schizo- phrenia-like psychosis [34] and b3gat1 knock-out mice have been reported to have synaptic plasticity defects [35,36]. Furthermore, GlcAT-P loss of function resulted in head morphogenesis defects in Medaka [37]. These studies together demonstrate a crucial role for GlcAT-P in nervous system development and/or function. However, despite the existence of a fly ortholog, very little is known about the role of GlcAT-P in the development and/or function of the Drosophila nervous system. Here, we describe the isolation and characterization of novel alleles of the GlcAT-P gene which we termed brave (brv). brv mutants are characterized by an elongated larval ventral nerve cord (VNC). We demonstrate that VNC elongation in brv mutants occurs during larval development and not during embryogenesis. We show that GlcAT-P is expressed in a subset of central and peripheral glia, and neurons in the larval CNS. We provide evidence that in brv mutant larvae the peripheral nerves are shorter as compared to wild-type and as a consequence, in GlcAT-P mutants, the VNC is possibly under mechanical tension and most likely responds by elongation along the anterior- posterior axis. This suggests a function of GlcAT-P in the growth of integrated peripheral nerves during larval development. We further demonstrate that for growth of peripheral nerves GlcAT- P is critically required in hemocytes. Finally, we identify the glial specific gene repo as a genetic modifier of GlcAT-P as the VNC elongation phenotype is dramatically enhanced if the repo gene dose is reduced in a brv mutant background. Thus, we suggest that GlcAT-P is a major component of the genetic network required in hemocytes and glia for stretch growth of integrated peripheral nerve fibers during Drosophila larval development. Our work is the first to identify a gene linked to the growth of peripheral nerves. GlcAT-P in Nervous System Development Results Isolation and genetic characterization of four novel GlcAT-P alleles To identify novel genes involved in larval brain morphogenesis, we carried out an EMS screen in the genetic background of DOPA- decarboxylase-GAL4 (DDC-GAL4); UAS-GFP transgenic flies. In these flies, the GFP reporter protein is localized in the cytoplasm of DDC-expressing cells, which allows easy visualization of the first (L1), second (L2) and third instar (L3) larval CNS [38]. Three pupal lethal mutants were isolated from the screen showing a dramatically 2–3 times extended larval ventral nerve cord (VNC) as compared to the VNC of wild-type controls (Figure 1A, B). In addition to the extension in the anterior-posterior axis, the mutant VNCs also displayed a considerable reduction in circumference. Moreover, the brain lobes in brv mutants were oval in shape (in the anterior-posterior direction), unlike their round morphology in wild-type. Genetically, these three mutations fell into one complementation group, demonstrating that they were indepen- dent alleles of the same gene, which we termed brave (brv). Accordingly, the three mutants were termed brv1, brv2 and brv3. By deletion mapping, we were able to assign a region on the third chromosome spanning 10 potential candidate genes (Figure 1C) to the brv mutants. By sequence analysis of both the genomic DNA and the cDNAs from those 10 genes, we could demonstrate that all three brv alleles were single point mutations in the GlcAT-P gene. In brv1, a G to A transition at the splice acceptor site located immediately before the start of the 7th exon resulted in a 14 bp deletion in the transcript RNA sequence due to a cryptic splicing site. As a result, a frame shift was introduced changing the amino acid sequence of the following 160 residues, before ending in a premature stop codon (see Figure 1D; Figure S1A, B). brv2 was found to be a G to A transition leading to a premature stop codon at the 10th amino acid of the 7th exon of GlcAT-P (Figure S1C). Finally, brv3 was characterized as a T to A transversion in the 27th position of the last (8th) exon resulting in a non-synonymous Leu to Gln polymorphism (conserved non-polar to polar amino acid) in this position (Figure S1D, E). To demonstrate that the observed brv mutant phenotype reflects a loss of GlcAT-P function and is not due to potential second site mutations, we followed two approaches: firstly, using the FRT containing transposons PBac{PB}CG14142c00688 and PBac{PB}c01618, we generated a GlcAT-P allele via flippase- induced precise deletion [39,40]. Precise deletion of GlcAT-P (henceforth referred to as Df(3L)GlcAT-P) was verified by PCR analysis and sequencing (data not shown). In addition, either complete depletion (for Df(3L)GlcAT-P), brv1 and brv2) or strong reduction (for brv3) of the GlcAT-P protein was demonstrated by immunoblotting using an anti-GlcAT-P antibody (Figure 1E). Df(3L)GlcAT-P homozygous larvae died during pupal stages and showed an extended larval VNC phenotype which was indistin- guishable from the other brv alleles. Moreover, Df(3L)GlcAT-P failed to rescue the brv1, brv2 and brv3 mutant alleles in complementation tests. These findings suggest that brv1, brv2 and brv3 are functional alleles. Secondly, a genomic construct containing exons 3 to 8 of GlcAT-P (P[acman] genomic construct CH322-82E19) rescued the larval brain defects and the pupal lethality of all three brv mutant alleles (Figure 1D). The dispensability of the upstream promoter region and the first two untranslated exons of GlcAT-P was surprising, but agreed with genetic evidence that a deficiency also lacking these elements (Df(3L)BSC379) genetically complemented the brv mutations (Figure 1D). This suggests that alternative functional promoters are present just upstream of the third exon of GlcAT-P. Although PLoS ONE | www.plosone.org 2 November 2011 | Volume 6 | Issue 11 | e28106