The glucuronyltransferase GlcAT-P is required for stretch growth of peripheral nerves in Drosophila

Abstract

During development, the growth of the animal body is accompanied by a concomitant elongation of the peripheral nerves, which requires the elongation of integrated nerve fibers and the axons projecting therein. Although this process is of fundamental importance to almost all organisms of the animal kingdom, very little is known about the mechanisms regulating this process. Here, we describe the identification and characterization of novel mutant alleles of GlcAT-P, the Drosophila ortholog of the mammalian glucuronyltransferase b3gat1. GlcAT-P mutants reveal shorter larval peripheral nerves and an elongated ventral nerve cord (VNC). We show that GlcAT-P is expressed in a subset of neurons in the central brain hemispheres, in some motoneurons of the ventral nerve cord as well as in central and peripheral nerve glia. We demonstrate that in GlcAT-P mutants the VNC is under tension of shorter peripheral nerves suggesting that the VNC elongates as a consequence of tension imparted by retarded peripheral nerve growth during larval development. We also provide evidence that for growth of peripheral nerve fibers GlcAT-P is critically required in hemocytes; however, glial cells are also important in this process. The glial specific repo gene acts as a modifier of GlcAT-P and loss or reduction of repo function in a GlcAT-P mutant background enhances VNC elongation. We propose a model in which hemocytes are required for aspects of glial cell biology which in turn affects the elongation of peripheral nerves during larval development. Our data also identifies GlcAT-P as a first candidate gene involved in growth of integrated peripheral nerves and therefore establishes Drosophila as an amenable in-vivo model system to study this process at the cellular and molecular level in more detail.

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 µm. (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: α-tubulin loading control.

Figure 2. Analysis of GlcAT-P expression as revealed by GlcAT-P-GAL4 driven reporter.

GlcAT-P is expressed in the larval brain lobes (A–G), the VNC (H–P) and peripheral nerves (Q–S) in the L3 larva brain. lacZ reporter expression in the brain lobes (red, A) colabeled with α-Repo (green, B). (C) Merged frames showing that GlcAT-P is expressed in a subset of Repo-positive glial cells (yellow; arrows). lacZ reporter expression in the mushroom bodies (red, D) colabeled with the neuronal marker Elav (green, E). Merged frame (F) showing co-staining of GlcAT-P and Elav in Kenyon cells (yellow; arrows). (G) Membrane targeted reporter (UAS-mCD8-mCherry) reveals expression of GlcAT-P in Kenyon cell neurites. lacZ reporter expression (H, K) in the larval VNC double labeled with α-Repo (I, J) or α-Elav (L, M). GlcAT-P is expressed in a subset of glial cells (yellow, arrows in J). GlcAT-P expression is also found in about two Elav positive neurons in ventral midline positions (yellow, arrows in M). (N–P) UAS-mCD8-mCherry reporter expression reveals that two cells (white arrow in N) bifurcate (arrowheads) and extend motor axons (blue arrows) bilaterally into the periphery. (P). GlcAT-P>lacZ reporter expression (Q) in cells on peripheral nerves are shown. These cells are peripheral glia as they colabel with Repo (R; yellow and arrows in S). A–F, H–J and K–M show individual frames of 1 µm thickness. G and N are maximum projections of Z-stacks. Bars in A, S: 50 µm. Bars in F, G: 10 µm.

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 µm.

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 immunostained 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 µm.

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 µm.

Figure 6. brv mutant larvae display shorter peripheral nerves.

Representative pictures of L1, L2 and L3 larval brains from brv⁺ (A, B, C) and brv¹ mutants (D, E, F) expressing Nrv2-GAL4;UAS-GFP^(S65T) to visualize the peripheral nerves. White arrowheads indicate the most posterior projecting and longest peripheral nerves used for these measurements. Histograms (G, H, I) compare the average lengths of the longest peripheral nerves during L1 (G), L2 (H) and L3 (I) larva between wild-type and brv¹ mutants. The peripheral nerves are significantly shorter in brv¹ mutants (*: p<0.0001). (J–S) Representative frames, taken from time-lapse recordings of wandering larva expressing Nrv2-GAL4; UAS-GFP^(S65T), display the VNC during one full cycle of a larval contraction and expansion during locomotion. A brv⁺ (J–N) and brv¹ larva (O–S) is shown. Ripples (arrowheads in P, Q), indicative for relaxation from tension, were detected only in VNC of brv¹ mutants but not in brv⁺ (J–N). Bar: 100 µm. A time line (in sec) is shown below panels J–S.

Figure 7. In brv mutant larvae the VNC appears to be under tension of peripheral nerves.

Representative pictures of L2 larvae from brv⁺ (A, B) and brv¹ mutants (C, D) expressing Elav-mCD8-GFP before (A, C) and after (B, D) dislodging the connections of the posterior most peripheral nerves. Arrowhead in (D) points towards a kink that appeared during relaxation of the VNC in brv¹ mutants. Representative pictures from brv⁺ (E), brv^1-/- (F), Tb^1-/+ (G) and brv^1-/-, Tb^1-/+ mutant (H) L3 larvae expressing Nrv2-GAL4;UAS-GFP^(S65T). The e\tension of the VNC in brv^1-/-, Tb^1-/+ is reduced as compared to brv^1-/-. Histograms (I, J) quantify the average lengths of VNCs and posterior-most peripheral nerves. brv^1-/-, Tb^1-/+ VNCs are significantly shorter as compared to brv^1-/- mutants (*: p = 0.0005). White arrowheads indicate the posterior tip of the VNC. Bars: 100 µm.

Figure 8. repo interacts genetically with GlcAT-P.

Representative pictures from brv⁺ (A), brv¹ (B), repo^-/+, brv^1-/- L3 (C) and repo^-/-, brv^1-/- L2 larvae. The CNS is visualized either by Nrv2-GAL4;UAS-GFP^(S65T) (A, B) or by repo-GAL4;UAS-GFP^(S65T) (C–D). White arrowheads indicate the tip of the VNC. (E, F) Histograms compare the average lengths of VNCs (E) and the most posterior peripheral nerves (F) for L3 as well as L2 larvae. repo^-/+, brv^1-/- VNCs are significantly longer as compared to brv^1-/- mutants (*: p<0.0001). Bar: 100 µm.