Neurofibromatosis-like phenotype in Drosophila caused by lack of glucosylceramide extension

Abstract

Glycosphingolipids (GSLs) are of fundamental importance in the nervous system. However, the molecular details associated with GSL function are largely unknown, in part because of the complexity of GSL biosynthesis in vertebrates. In Drosophila, only one major GSL biosynthetic pathway exists, controlled by the glycosyltransferase Egghead (Egh). Here we discovered that loss of Egh causes overgrowth of peripheral nerves and attraction of immune cells to the nerves. This phenotype is reminiscent of the human disorder neurofibromatosis type 1, which is characterized by disfiguring nerve sheath tumors with mast cell infiltration, increased cancer risk, and learning deficits. Neurofibromatosis type 1 is due to a reduction of the tumor suppressor neurofibromin, a negative regulator of the small GTPase Ras. Enhanced Ras signaling promotes glial growth through activation of phosphatidylinositol 3-kinase (PI3K) and its downstream kinase Akt. We find that overgrowth of peripheral nerves in egh mutants is suppressed by down-regulation of the PI3K signaling pathway by expression of either dominant-negative PI3K, the tumor suppressor PTEN, or the transcription factor FOXO in the subperineurial glia. These results show that loss of the glycosyltransferase Egh affects membrane signaling and activation of PI3K signaling in glia of the peripheral nervous system, and suggest that glycosyltransferases may suppress proliferation.

Fig. 1.

egh mutants exhibit enlargement of peripheral nerves with attached immune cells. (A) Biosynthesis of glycosphingolipids in Drosophila. (B, C, and E) Bright-field photomicrographs of the nervous system in dissected third instar larvae. Dorsal view; anterior is upward. The bilateral nerves innervating abdominal segment 9 (A9) including their exit from the ventral nerve cord (VNC) are in focus. Other nerves originating from more anterior segments are also visible. Brackets indicate the diameter of the right A9 nerve in wild type (B) and egh^62d18 (C). Note in C the ∼50% increase in nerve diameter, and the plasmatocytes (PC) accumulating on the nerves. (D and D′) Expression of the plasmatocyte-specific marker eater-GFP in the egh background (egh^62d18/Y; eater-GFP/+) verifies the identity of the nerve-attached cells in egh as plasmatocytes. (E) Rescue of the egh phenotype by ubiquitous expression of an Egh transgene (egh/Y; UAS-Egh/+; act5c-Gal4/+). [Scale bar, 50 μm (B–E).] (F) Scatter plot of the diameter of the A9 nerves, measured 48 μm from the VNC exit. Data are mean ± SEM; **P < 0.001, ***P < 0.0001. (G) The proportions of larvae with plasmatocyte accumulation on A9 nerves. Error bars mark 95% confidence intervals.

Fig. 4.

Hypertrophic nerves with attached immune cells in is due to lack of GlcCer extension. (A) Biosynthesis of GlcCer-related glycosphingolipids in vertebrates. (B and C) Bright-field photomicrographs of larval peripheral nerves near the VNC. Brackets indicate the diameter of the right A9 nerve in (B) brn^1.6P6 and (C) after introduction of mammalian β4GalT6 (egh/Y; Gli-Gal4/UAS-β4GalT6). Note rescue of the egh phenotype in C. [Scale bar, 50 μm (B and C).] (D) Scatter plot of the A9 nerve diameter. Mean ± SEM is indicated; ***P < 0.0001. n.s., not significant. (E) Proportions of larvae with plasmatocyte accumulation on A9 nerves. Error bars mark 95% confidence intervals. The egh^62d18 allele was used in C–E.

Fig. 2.

egh mutants have disorganized peripheral nerves with an increased number of subperineurial glial cells. (A) TEM micrographs of cross-sections of larval A9 nerves from wild type (Left) and egh^62d18 (Right), at identical magnification. The egh image was obtained using MIA. Axons, wrapping glia, subperineurial glia, and perineurial glia layers are indicated on the wild-type nerve. In the egh nerve only the axons are colored, because it was not possible to discern the glial subtypes. (B) TEM MIA micrographs exemplifying the most severe egh nerve phenotype, with a nerve diameter of 16 μm, and tightly encapsulated by attached plasmatocytes (asterisks in B and B′). Higher magnification in B′ of outlined area shows plasmatocyte-mediated deposition and/or breakdown of the outermost layer of the nerve, the extracellular matrix (plasmatocytes are marked by asterisks). (C and D) Confocal light micrographs of cross-sections reconstructed from Z-stack scans of individual peripheral nerves. Glial cell nuclei were visualized by anti-Repo (red) and subperineurial glia by Gli-Gal4/UAS-GFP (green) in wild-type (C) or egh mutant (D) background. (E and F) Intensity-inverted fluorescence light micrograph of subperineurial glial cell nuclei visualized by Gli-Gal4/UAS-GFPnls. Green stars indicate individual nuclei in wild-type (E) or egh background (F). (G) Number of subperineurial glial (SPG) cell nuclei per A9 nerve, visualized as in E and F. For both wild type and egh, data were obtained from six larvae. Data are mean ± SEM; ***P < 0.0001.

Fig. 3.

PI3K signaling is required for manifestation of the egh nerve phenotype. (A–F) Bright-field light microscopy images of larval nerves near the VNC. Brackets indicate the diameter of the right A9 nerve. (A) Gli-Gal4/UAS-Ras^V12; (B) egh/Y; Gli-Gal4/UAS-Ras^V12; (C) UAS-Dp110^CAAX/Y; Gli-Gal4/+; (D) egh/Y; Gli-Gal4/UAS-Dp110^D954A; (E) egh/Y; Gli-Gal4/UAS-PTEN; (F) egh/Y; Gli-Gal4/UAS-FOXO. [Scale bar, 50 μm (A–F).] (G) Scatter plot of the A9 nerve diameter 48 μm posterior to the VNC. Data are mean ± SEM; **P < 0.001, ***P < 0.0001. n.s., not significant.