Figure 4. Parasegmental identity and sequence of homeotic gene expression domains are not altered in brv mutants. L3 larva brains from brv+ (A–D) and brv1 mutants (E–H) were immuno- stained with anti-Antp (A, E), anti-Ubx (B, F), anti-AbdA (C, G), and anti- AbdB (D, H). (I–L) Histograms comparing the average lengths of the homeotic expression domains (I: Antp; J: Ubx; K: AbdA; L: AbdB) are shown. Dashed lines indicate parasegmental (PS) boundaries. Bar: 20 mm. doi:10.1371/journal.pone.0028106.g004 elongated VNC. To resolve this issue, time lapse recordings of wandering L3 wild-type and brv mutant larvae were performed. In wild-type larvae, the brain and VNC as well as the peripheral nerves were rapidly stretched and relaxed as the larvae moved (Figure 6J–N; see also [53]). However, stretching and compression was more evident in brv mutant larvae, in which the VNC contracted more strongly and developed ripples as the larvae constricted (arrowheads in Figure 6O–S). This suggested that the larval VNC was under higher tension in brv mutants. To further investigate whether the VNC is indeed under tension of shorter peripheral nerves, we made use of elav-mCD8-GFP transgenic flies [48] and observed the VNC in real time in wild-type and brv mutant L2 larvae. Larvae were cold-immobilized and a cut was made at the posterior end. This cut severed the connections between the most posterior peripheral nerve fibers and their respective body wall contacts and as a consequence any tension of the most posterior nerves on the VNC would be released. We observed that after this cutting procedure the VNC immediately retracted in brv mutants (n = 10; Figure 7C, D), whereas there were no detectable changes in wild-type larvae (n = 10; Figure 7A, B). VNC retraction could not be seen in brv mutant L3 larvae after an identical procedure, possibly because during L3 larval stages the VNC is too deformed to return to its original length. To further resolve if VNC elongation is the cause or the consequence of shorter peripheral nerves, we made use of the Tubby (Tb) mutant. Tb larvae are considerably shorter along the anterior-posterior axis than wild-type larvae [13]. Although the length of the Tb VNC was GlcAT-P in Nervous System Development comparable to wild-type (Figure 7G, I), peripheral nerves were shorter in Tb larvae (Figure 7G,J). However, when we compared VNC extension in brv-/- and in brv-/-; Tb-/+ mutant L3 larvae, we found that in brv-/-; Tb+/- mutants the VNC was also extended but significantly lesser as compared to brv-/- mutants alone (Figure 7E– I). This experiment demonstrated that the extension of the VNC in brv mutants correlates with the total length of the larva. Since the peripheral nerves and the tracheal system are the only physical connections between the larval body wall and the CNS, our results suggested that in brv mutants the VNC expands as a consequence of increased tension from shorter peripheral nerves or trachea possibly due to some growth retardation of larval peripheral nerves or trachea. GlcAT-P function is required in hemocytes To test for the cell specific requirement of GlcAT-P, rescue experiments with ubiquitous (Act5C-GAL4) as well as cell specific GAL4 drivers for the tracheal system (btl-GAL4), neuronal (Elav- GAL4) and glial cells (gcm-GAL4 and repo-GAL4) were performed. We were unable to rescue brv mutants with the btl-GAL4 tracheal driver ruling out any involvement of the tracheal system at least in the VNC extension phenotype. We also could not rescue brv mutants with the pan-neuronal driver Elav-GAL4 demonstrating that expression of GlcAT-P in neurons is not sufficient to rescue the VNC extension phenotype. However, ubiquitous expression by Act5C-GAL4 completely rescued the phenotype (Figure S7; n = 50) showing that the VNC extension in brv mutants is a specific consequence of loss of GlcAT-P function. Furthermore, the catalytically dead version of GlcAT-P (GlcAT-Pcd) driven by Act5C-GAL4 failed to rescue demonstrating that impairment of GlcAT-P catalytic activity is sufficient for the brv mutant phenotype. GlcAT-P driven by the pan-glial and hemocyte driver gcm-GAL4 fully rescued the extended VNC phenotype as well as the pupal lethality (Figure S7; n = 50). However, another pan-glial driver, repo-GAL4, was not able to rescue the brv mutant phenotype (Figure S7; n = 50). Most importantly, the brv mutant phenotype was completely rescued (Figure S7; n = 50) when GlcAT-P was driven by the hemocyte specific driver, crq-GAL4. The rescue experiments identified a critical role for GlcAT-P in hemocytes. repo is a genetic modifier of GlcAT-P In an attempt to identify interactors with GlcAT-P, we tested a possible genetic interaction with the glial specific repo gene. Our first observation that repo and GlcAT-P interacted came from our rescue experiments when we observed enhanced elongation of the VNC in the presence of one chromosome carrying the lethal repo- GAL4 (henceforth referred to as repo) insertion in the background of brv1-/- larvae (Figure S7). The length of the extended VNC in brv1-/-, repo-/+ L3 larvae was 560.8+/- 44.8 mm as compared to 382.8+/- 54.8 mm in brv1-/- mutant larvae (Figure 8A–C, E; n = 7 for each). This difference was statistically significant (p,0.0001). Correspondingly, the posterior-most peripheral nerves shortened from 508.6+/- 40.7 mm in L3 brv1-/- mutant larvae to 283.3+/- 48.8 mm in brv1-/-, repo-/+ larvae (Figure 8A–C, F; n = 7 for each). The shortening of peripheral nerves is statistically significant (p,0.0001). Furthermore, rare brv1-/-, repo-/- L2 escapers showed an even more severe extension of the VNC as compared to brv-/- L2 larvae and a similar extension as brv-/-, repo-/+ L3 larvae (Figure 8D, E). The increase in brv1-/-, repo-/- L2 larvae was also significant (p,0.0001) compared to L2 brv-/- larvae. It is worth noting that the VNC of brv1-/-, repo -/- L2 larvae almost spanned the entire length of the larva (Figure 8E; see also Figure 3L), a situation similar to late embryos where the VNC extends to the most posterior end of the embryo. Interestingly, the peripheral PLoS ONE | www.plosone.org 7 November 2011 | Volume 6 | Issue 11 | e28106