GlcAT-P in Nervous System Development Figure 5. Larval VNC extension is not a consequence of extra larval NB and GMC proliferation. (A–C) Comparison of progenitor cells (neuroblasts and GMCs) in the Antp expressing domain (anti-Antp staining not shown) of (A) brv+ and (B) brv1 mutants using anti-Mira. (C) Quantification of Mira positive cells in brv+ and brv1. (D–I) anti- Nrt staining in thoracic (D–F) and abdominal neuromeres (G–I) of L3 larva brains in brv+ (D, G) and brv1 mutants (E, F, H,I). A1–A9: abdominal neuromeres 1 to 9. Arrowheads in H point to the 3 Nrt positive NB lineages in A3. A–B and D–I represent maximum projections of Z-stacks. Bar: 20 mm. doi:10.1371/journal.pone.0028106.g005 nerves were also severely shorter in L2 larvae suggesting that growth of peripheral nerves came to an almost complete halt in brv1-/-, repo-/- double mutants (Figure 8F). These results strongly support the notion that hemocytes as well as glial cells are synergistically required for the growth of integrated peripheral nerves during larval development. Taken together, our data demonstrate that VNC extension in brv mutants may be a result of increased tension exerted on the VNC as a consequence of impaired peripheral nerve extension/ growth. In this context, GlcAT-P plays a crucial role in hemocytes which might control some aspect of glial cell biology implicated in the growth of integrated peripheral nerves. Further studies are required to address the exact molecular and cellular mechanisms of GlcAT-P function in hemocytes and glia. Discussion In this study, we have identified and characterized novel alleles of the glucuronyltransferase GlcAT-P, the Drosophila homolog of human B3GAT1. The loss of GlcAT-P function causes an extension of the VNC probably due to the inability of the peripheral nerve to stretch/grow in tandem with the growth of the larval body. We also showed that hemocytes are critically required for the extension of peripheral nerves during larval development but glia also plays an important role. Our findings suggest that GlcAT-P is involved in growth of integrated peripheral nerves during Drosophila larval growth. GlcAT-P is expressed in the larval nervous system Although we spent considerable effort to study GlcAT-P protein localization in larval brains, we were unsuccessful in detecting any relevant signal using an antibody raised against Drosophila GlcAT-P in tissue whole mounts although the antibodies worked on western blots and in an over/mis-expression paradigm. The fact that we could not detect GlcAT-P expression in specimen could be attributable to a low endogenous expression level of GlcAT-P or, alternatively, to steric hindrance of the antigenic epitope(s) due to the particular subcellular localization of the GlcAT-P protein or to its interaction with other proteins. However, a GlcAT-P-GAL4 line with an insertion into the GlcAT-P locus allowed us to study the embryonic and larval expression pattern of GlcAT-P. GlcAT-P- GAL4 driven LacZ reporter expression could not be detected in the hemocytes or embryonic CNS. But LacZ expression was clearly detected in a subset of neurons (Kenyon cells and VUM PLoS ONE | www.plosone.org 8 November 2011 | Volume 6 | Issue 11 | e28106