GlcAT-P in Nervous System Development Figure 3. The VNC in brv mutants elongates during larval development and can be rescued by ectopic GlcAT-P expression. An Elav- mCD8-GFP background is used to visualize the outlines of the VNC. (A–C) Representative images of stage 17 embryos. (A) Wild-type (brv+) and (B) brv1 maternal and zygotic mutant. The progressive elongation of the VNC during larval development is shown in L1 (E–G), L2 (I–K) and L3 (M–O). Larval brains from wild-type (E, I, M), brv1 (F, J, N) and specimen over-expressing GlcAT-P (C, G, K, O) in brv1 mutant background are shown. (D, H, L, P) Quantification of the length of the VNC (n = 7, for each stage) is presented (* refers to CH322-82E19 rescued specimen. Double-sided arrows indicate the length of the VNC. L1, 2, 3 refers to larval stages 1, 2, and 3. Bars: 50 mm. doi:10.1371/journal.pone.0028106.g003 stainings (Figure S4). We did not find any major changes in the expression pattern of these genes and we concluded that the structural integrity of the VNC was not grossly affected in brv mutants. We also checked the integrity of mushroom body neurons and VUM neurons in brv mutant larval brains by labeling them with UAS-mCD8-GFP using GAL4-OK107 [50] and 807-GAL4 (A. Brand, unpublished), respectively. The brv mutation did not affect the viability of these neurons, but a displacement of their cell bodies and neurites along the longitudinal axis was observed (Figure S5) possibly as a consequence of the elongation of the nervous system along the anterior-posterior axis. Neuroblast proliferation is normal in brv mutants To examine whether the extension of the VNC could be due to additional cells, we studied neuroblast (NB) proliferation in brv mutants. We firstly analyzed the number of proliferating cells in thoracic and abdominal segments of brv mutant VNCs during L3 larvae by anti-Miranda staining. Miranda (Mira) labels dividing NBs and ganglion mother cells (GMCs). We could not detect any significant difference in the number of proliferating Mira positive cells in brv mutant versus wild-type larval brains (n = 6; Figure 5A– C). We also analyzed neuroblast lineages in the larval VNC of L3 brv mutants using anti-Neurotactin (Nrt) immunohistochemistry. In L3 larva, Nrt labels active NBs and their larval-specific secondary neurons [51]. Extra Nrt positive cells indicative of extra proliferation could not be detected in thoracic segments (Figure 5D–F) or in abdominal segments (Figure 5G–I) of the VNC. For example, in wild-type VNCs, only 3 NBs per hemisegment are mitotically active during L3 stages producing small lineages that contain 4 to 12 Nrt positive cells [52]. Both the number of Nrt labeled NB lineages and the number of cells within these lineages was comparable between wild-type and brv mutant abdominal neuromeres (Figure 5G, H–I). However, as a consequence of the VNC extension, the spacing between the abdominal Nrt positive lineages was increased in brv mutant larvae (Figure 5H, I). In the thoracic segments, the extension resulted in a misplacement of NB lineages towards the midline of the VNC, a domain reserved to the neuropile in wild-type brains. In summary, we present evidence that the elongation of the VNC in brv mutant larvae is not due to extra cell proliferation of larval NBs. Peripheral nerves are shorter in brv mutant larvae Since GlcAT-P-GAL4 reporter gene expression was found in the peripheral nerves and in the VUM motoneurons whose axons project through the peripheral nerves towards their peripheral muscle targets (Figure 2K, N, Q), we investigated whether the length of these peripheral nerve fibers was affected in brv mutant larvae. Using Nervana2-GAL4 (Nrv2-GAL4); UAS-GFP transgenic flies [53], we measured the length of the longest nerve fibers emanating from the most posterior end of the VNC in brv mutants and compared it to wild-type. A substantial difference in the length of the peripheral nerves was already detectable during L1 stages but not at stage 17 embryos (data not shown) and the difference was more evident in L2 and L3 larva (Figure 6A–I). We found that nerve fibers in brv mutants were considerably shorter (wild-type: 750+/- 36 mm; brv mutants: 508+/- 40 mm, n = 7 for each) (Figure 6C, F, I) at L3 stage. The data demonstrated that consistently peripheral nerves were shorter by about one third in brv as compared to wt. As a consequence of the nerve fibers being shorter, they also contained less total peripheral glia as compared to wild-type, although the spacing and the distribution of peripheral nerve glia on a similar long stretch of the nerve fibers did not seem to be affected (Figure S6). Shorter peripheral nerves could either be causative for the extension of the VNC or, alternatively, a consequence of the PLoS ONE | www.plosone.org 6 November 2011 | Volume 6 | Issue 11 | e28106