mutant larvae altered ECM deposited by GlcAT-P deficient hemocytes and glia might lead to profound changes in the ECM to cell signaling resulting in the observed peripheral nerve growth phenotype. It is conceivable that ECM related mechanisms contributing to early growth cone pathfinding are also involved in growth of axons after the growth cones reached their final target sites. However, currently the precise molecular and cellular mechanisms how GlcAT-P affects axonal extension of integrated axons in Drosophila peripheral nerves are unknown and need further investigations. Alternatively, GlcAT-P could play a role in glial proliferation or migration. This finds some support by the observation that at least the peripheral nerves had less total glia although spacing and distribution of glial cells along the nerves seemed to be unperturbed. The limited reduced number of peripheral glia could then hamper the extension of peripheral nerves and thus limit the elongation of peripheral axons. However, we did not observe a general reduction of glial cells in brv mutants making a general role of GlcAT-P in glial proliferation relatively unlikely. In summary, we have isolated novel alleles of the Drosophila homolog of the mammalian glucuronyltransferase b3gat1, GlcAT-P. Functional analysis suggests that GlcAT-P is required in hemocytes for the growth of peripheral nerves during development in which glial cells also play a major role. The postembryonic growth of organisms requires the continuous elongation of integrated peripheral nerves and axons to accommodate for the extension of the body axes during development. To our knowledge, GlcAT-P is the first gene with a possible link to nerve stretch growth. We propose that the developing Drosophila larval nervous system and the GlcAT-P mutants characterized here will be helpful in deciphering the basic cellular and molecular mechanisms of nerve stretch growth in the future. Currently, the major challenge is the identification of potential GlcAT-P targets either via biochemical or genetic screens and the characterization of their functional involvement. Given the high degree of functional conservation of molecular and biochemical pathways between insects and vertebrates, performing further mechanistic studies on GlcAT-P in Drosophila most likely will deliver new insights into the roles of glucuronyltransferases in brain develop- ment and function. Materials and Methods Fly stocks w*; DDC-GAL4 [38] and w*; UAS-GFPS65T were meiotically recombined on the third chromosome. For the EMS mutagenesis screen w*; DDC-GAL4, UAS-GFPS65T males were fed with 25 mM EMS according to standard procedures. Following this, they were mated with w*; Dr1/TM6b, Dfd-GMR-nv-YFP, Sb1, Tb1 virgin females. Single males from the progeny were then mated with w*; Dr1/TM6b, Dfd-GMR-nv-YFP, Sb1, Tb1 virgin females to propagate the mutagenized third chromosome. Finally, w*; DDC-GAL4, UAS- GFPS65T #/Dr1/TM6b, Dfd-GMR-nv-YFP, Sb1, Tb1 males and female virgins were mated together to establish the stocks (# denotes mutagenized chromosome). DDC-GAL4, UAS-GFPS65T was meiotically recombined out of brv1, brv2 and brv3 mutant background. The following fly strains were also used: elav-mCD8-GFP [48], FRT2A, UAS-mCD8-GFP, dppblink-GAL4 [78], Sqh::EYFP- Golgi [43], UAS-nbgal, hs-Flp, P{GawB}eyOK107, PBac{GAL4D, EYFP}GlcAT-PPL00294 (used at GlcAT-P-GAL4), PBac{PB} CG14142c00688, PBac{PB}c01618, 807-GAL4 (A Brand, unpub- lished) and Tb1. Fly stocks without reference are described in flybase. Unless otherwise specified, all the mutant analysis was GlcAT-P in Nervous System Development done in brv1 mutant allele. GlcAT-P cDNA was amplified from DGRC plasmid LD40245 by PCR using the oligos RP1 (AATTGCGGCCGCTTGGTTCGTCACAATTTTTTATA) and RP2 (AATTGGTACCTTTTTTTTTTTTTTTTTTGGAAAA- TG). Catalytically dead GlcAT-P was generated by mutating Asp333 to Ala by PCR. For making respective UAS-GlcAT-P lines, the PCR products were cloned into the NotI-KpnI cleaved transformation vector pUAST-attB. Mapping the EMS lesions RNA was extracted from homozygous brv mutant larvae using the RNAeasy kit (Qiagen). cDNAs derived from the 10 genes in the mapped region were synthesized using standard protocols (Invitrogen) and sequenced for all the three alleles. Rescue constructs and EGFP tagging by recombineering The following construct from P[acman] resource (see http:// flypush.imgen.bcm.tmc.edu/pacmanfly) was used for rescue experiments: CH322-82E19 (contains GlcAT-P without the first two exons). N or C terminus of GlcAT-P was tagged to EGFP in CH322-82E19 and CH321-38B20 (contains entire GlcAT-P and some other genes) BACs from P[acman] resource. We used Addgene plasmids 19173 and 19178 for EGFP-tagging by recombineering [45]. All germ line transformants were generated by Bestgene Inc, CA. Antibody generation Polyclonal antibodies were generated against peptides C- RTRYKNTNLEHIDRLLVRP and C-EGRNALISKNGREN- PHSK for GlcAT-P and GlcAT-S respectively (Genzym Antibodies). Immunoblotting Third instar larvae or their brains were used for extract preparation. Protein extracts were resolved by SDS-PAGE. Immunoblots were probed with anti-HNK-1 (1:1000), rabbit anti-GlcAT-P (1:1000), rabbit anti-GlcAT-S (1:1000) and mouse anti-a-tubulin (1:10000, Sigma) using PVDF membranes (Perkin Elmer) and Supersignal-detection kit (Thermo Scientific). Immunohistochemistry The following primary antibodies were used: mouse anti-elav (1:20, Developmental Studies Hybridoma Bank (DSHB)), mouse anti-repo (1:5, DSHB), mouse anti-antp (1:20, DSHB), mouse anti- AbdB (1:10, DSHB), mouse anti-Ubx (1:20, kindly provided by R White), rat anti-AbdA (1:200, kindly provided by J Casanova), mouse anti-HNK-1 (1:1000, kindly provided by S Oka), rabbit anti-Mira (1:5000, kindly provided by F Matsuzaki). In addition, secondary goat antibodies conjugated to Alexa488 (Molecular Probes), Cy3 or Cy5 (Jackson Immuno Research Laboratories) were applied. Images were acquired with an Upright Olympus FV-1000 Confocal Imaging system using FV10-ASW software. For releasing the connections of the posterior peripheral nerves with the larval muscles, the appropriately staged wild-type and brv mutant larvae carrying elav-mCD-GFP were first cold-immobilized. An image was captured using Leica MZ16F stereomicroscope before and after the posterior-most nerve connections were separated by making a minute cut in the posterior part of the larvae. Supporting Information Figure S1 Molecular characterization of brv1, brv2 and brv3 alleles. Partial alignments of the cDNA (A, C, D) and genomic PLoS ONE | www.plosone.org 12 November 2011 | Volume 6 | Issue 11 | e28106