GlcAT-P in Nervous System Development Figure 1. brv mutations are novel alleles of Drosophila GlcAT-P. (A, B) Phase contrast pictures of wild-type (brv+) and brv1 mutant L3 larva brains showing the elongation of the larval VNC in brv (B). Bar: 50 mm. (C) Graphical display of the deficiencies (Df) used in the complementation test in which the chromosomal region was narrowed to 60 kb. (+), (-) indicate complementing and non-complementing Df, respectively. (D) Illustration of the structure of the GlcAT-P locus flanked by the predicted genes CG14142 and CG6199. Arrows indicate the direction of transcription. Exons are boxed and translated regions are represented by filled boxes. The gray box indicates an exon which is spliced into one but not the other isoform. The positions in which the brv1, brv2 and brv3 alleles are mutated are indicated. (E) Analysis of GlcAT-P protein expression. Protein extracts from wild-type (brv+), Df(3L)GlcAT-P, brv1, brv2 and brv3 mutant L3 larvae were blotted with an anti-GlcAT-P antibody. Tub: a-tubulin loading control. doi:10.1371/journal.pone.0028106.g001 all 4 brv mutant alleles are generally pupal lethal, we found adult escapers (,10%, n = 200) for all alleles. The escapers almost always showed developmental defects in the legs (,98%, n = 100) and some had rudimentary wings (,30%, n = 100). GlcAT-P is contributed maternally (see BDGP; http://www. fruitfly.org/cgi-bin/ex/bquery.pl?qtype=report&find=CG6207 &searchfield=CG). To address the function of maternally expressed GlcAT-P, we generated brv1 germline clones using the FLP-DFS technique [41]. We could not detect an abnormal phenotype in the nervous system of embryos lacking both maternal and zygotic GlcAT-P (data not shown; see below), indicating that this gene is dispensable for embryonic nervous system development. GlcAT-P is a key enzyme for HNK-1 epitope biosynthesis in the nervous system [42]. In mice, depletion of GlcAT-P results in an almost complete loss of HNK-1 expression in the brain [35]. On the contrary, Drosophila Df(3L)GlcAT-P larvae showed a similar pattern of HNK-1 modified proteins on immunoblots of brain lysates when compared to wild-type (Figure S2A). To test for possible redundancy of GlcAT-P and GlcAT-S, an anti-GlcAT-S antibody was used in immunoblots of protein extracts from Df(3L)GlcAT-P larval brains. GlcAT-S-specific bands were clearly detected (Figure S2A). Thus, functional redundancy of GlcAT-P and GlcAT-S might explain the presence of HNK-1 in Df(3L)GlcAT-P larva. GlcAT-P is expressed in a subset of glia, VUM motoneurons and mushroom body neurons To study the expression pattern of GlcAT-P during larval development, we raised an anti-GlcAT-P antibody. However, we did not obtain specific signals in whole mount L3 larval brains using standard immunohistochemistry. To evaluate the function- ality of the antibody, we performed immunolabeling with wing imaginal discs from transgenic larvae carrying dppblink-GAL4 and UAS-GlcAT-P. A clear signal was detected along the anterior posterior border of the wing disc (Figure S2B) which was in accordance with the endogenous dppblink-GAL4 expression pattern. Using an EYFP-tagged Golgi-specific protein (Sqh::EYFP-Golgi, [43]), we detected the over-expressed GlcAT-P protein in the Golgi apparatus (Figure S2C), where GlcAT-P is normally localized [44]. These findings indicate that the antibody specifically recognizes GlcAT-P protein in an over-expression paradigm in the right cellular compartment. As an alternative approach, we generated N-terminal as well as C-terminal EGFP- tagged fusion constructs of GlcAT-P by recombineering [45], using P[acman] genomic constructs containing the entire GlcAT-P locus (see methods). However, neither could we detect any specific EGFP signal in immunohistochemistry experiments, nor did the fusion constructs rescue the brv mutants, indicating that the EGFP tagging impairs GlcAT-P sub-cellular localization and/or func- tion. We also attempted in situ hybridization experiments with GlcAT-P probes in the larval brain. However, although the probe was shown to work specifically when GlcAT-P was over-expressed in wing discs (data not shown), any in situ signal above background was not observed in the larval brain. Lastly, we made use of a GAL4-containing transposon inserted at the GlcAT-P locus (Figure 1D, henceforth referred to as GlcAT-P-GAL4). When used to drive expression of the reporter genes UAS-nbgal or UAS-mCD8- mCherry, GlcAT-P-GAL4 showed expression in the salivary gland primordia, amnioserosa and gut during embryogenesis (Figure S3A, B; data not shown), mirroring most aspects of the endogenous in situ hybridization expression pattern (see BDGP; http://www.fruitfly.org/cgi-bin/ex/bquery.pl?qtype=report& find =CG6207&searchfield=CG). This indicates that GlcAT-P- GAL4 recapitulates the endogenous expression pattern of GlcAT-P. In the larva, reporter expression was detected in the tracheal system which connects to the VNC (data not shown). GlcAT-P-GAL4 drove reporter gene expression in the nervous system in sets of neurons in the brain lobes (Figure 2A– G), the VNC (Figure 2H–P) as well as in the peripheral nerves emanating from the VNC (Figure 2Q–S). Reporter gene expression was detected in a subset of glial cells in the brain and the VNC. In addition, GAL4 was expressed in most, but not all, of the peripheral nerve glia. Within the larval brain lobes, GAL4 was also detected in mushroom body neurons (Figure 2D–G). In the VNC GlcAT-P-GAL4 expression was also found in two VNC neurons per neuromere (arrows in Figure 2M, N). Based on their location in ventral positions of the ventral midline, their bifurcating axons (arrowheads in Figure 2N) leaving the VNC through the peripheral nerves (red arrows in Figure 2N) and the transcriptional activity of a VUM-specific GAL4 line (807-GAL4, see Figure S5), these neurons were characterized as ventral-unpaired-median (VUM)-motoneu- rons [46,47]. In brv mutants the VNC is elongated during larval development To determine when the VNC elongation occurs developmen- tally, we examined central nervous system development during embryogenesis and larval stages in brv mutants in the background of elav-mCD8-GFP transgenic flies [48]. During late embryogenesis (stage 17), elongation of the VNC could not be detected in maternal and zygotic brv mutants (Figure 3A–D). However, in brv mutant L1 larvae, an abnormally extended VNC was readily detectable (n = 7; Figure 3E–H). As larval development progressed to L2 (n = 7; Figure 3I–L) and L3 (n = 7; Figure 3M–P), the elongation of the VNC became more pronounced. We thus conclude that VNC elongation in brv mutants does not take place during embryonic development, but starts during early larval development and progresses through L2 and L3 larval stages. To test if the sequence as well as the size of metameric units in the L3 VNC were affected in brv mutants, we analyzed the expression profile of the homeotic genes Antp, Ubx, abdA and abdB, which have been described to label parasegments PS3–PS5, PS5– PS6, PS7–PS12 and PS13–PS14, respectively [49]. Our results showed that all the parasegments in the brv mutants were present and in the correct sequence (Figure 4) suggesting that the homeotic identity of the parasegments as well as their sequence was correctly specified in the larval VNC of brv mutants. We checked the intactness of the VNC in brv mutant larvae by analyzing anti- Futsch/22C10, anti-BP102, anti-Fas2, anti-Elav and anti-Repo PLoS ONE | www.plosone.org 4 November 2011 | Volume 6 | Issue 11 | e28106