A versatile genetic tool to study midline glia function in the Drosophila CNS

Abstract

Neuron-glial interactions are crucial for growth, guidance and ensheathment of axons across species. In the Drosophila CNS midline, neuron-glial interactions underlie ensheathment of commissural axons by midline glial (MG) cells in a manner similar to mammalian oligodendrocytes. Although there has been some advance in the study of neuron-glial interactions and ensheathment of axons in the CNS midline, key aspects of axonal ensheathment are still not fully understood. One of the limitations has been the unavailability of MG membrane markers that could highlight the glial processes wrapping the axons. Previous studies have identified two key molecular players from the neuronal and glial cell types in the CNS midline. These are the neuronal transmembrane protein Neurexin IV (Nrx IV) and the membrane-anchored MG protein Wrapper, both of which interact in trans to mediate neuron-glial interactions and ensheathment of commissural axons. In the current study, we attempt to further our understanding of MG biology and try to overcome some of the technical difficulties posed by the lack of a robust MG driver that will specifically allow expression or knockdown of genes in MG. We report the generation of BAC transgenic flies of wrapper-GAL4 and demonstrate how these flies could be used as a genetic tool to understand MG biology. We have utilized the GAL4/UAS system to drive GFP-reporter lines (membrane-bound mCD8-GFP; microtubule-associated tau-GFP) and nuclear lacZ using wrapper-GAL4 to highlight the MG cells and/or their processes that surround and perform axonal ensheathment functions in the embryonic midline. We also describe the utility of the wrapper-GAL4 driver line to down-regulate known MG genes specifically in Wrapper-positive cells. Finally, we validate the functionality of the wrapper-GAL4 driver by rescue of wrapper mutant phenotypes and lethality. Together, these studies provide us with a versatile genetic tool to investigate MG functions and will aid in future investigations where genetic screens using wrapper-GAL4 could be designed to identify novel molecular players at the Drosophila midline and unravel key aspects of MG biology.

Keywords: Axonal ensheathment; Commissures; Midline glia; Neurexin IV; Wrapper; wrapper-GAL4.

Figure 1. Generation and expression of wrapper-GAL4

(A-C) Confocal images of Stage 16 embryos of sim-GAL4/UAS-tauGFP (A), slit-GAL4/UAS-tauGFP (B) and wrapper-GAL4/UAS-tauGFP (C, Janelia GAL4 stock abbreviated as JF) show expression of GFP (green) and BP102 (red). (D) Design of the wrapper-GAL4 construct using BAC CH321-61P08. The DNA sequence in blue is from the wrapper gene with its initiator codon ATG shown in red. The sequence in green is the GAL4 sequence and the termination codon is shown in green with asterisk. Further downstream the wrapper sequence continues in blue. No wrapper sequences were deleted but only disrupted with inframe insertion of GAL4. (E) Confocal image of Stage 16 embryo of wrapper-GAL4/UAS-mCD8-GFP in ventral view shows GFP expression in MG (E) and BP102 in CNS axons (E′). (F-F″ and G-G″) Higher magnification confocal images of Stage 16 embryos of wrapper-GAL4/UAS-mCD8-GFP (F-F″, ventral view, and G-G″ lateral view) shows an overlap of Wrapper with GFP (F″ and G″). (H-H″ and I-I″) Confocal images of Stage 16 wrapper-GAL4/UAS-tau-GFP embryos in ventral view (H-H″) and lateral view (I-I′) also display an overlap of Wrapper (H and I) with GFP (H′ and I′) at the MG. GFP expression can also be seen in glial processes (green asterisk, H′). BP102 (blue) was used for labeling CNS axonal scaffold. Embryos in A, B and C show a lateral view and are oriented anterior to the left and dorsal top. Embryo in E shows a sagittal view with anterior to the left. Higher magnification images in F-I″ have anterior to the top. (J-J‴) Confocal images of MG and their ensheathing processes in third instar ventral nerve cord of wrapper-Gal4/UAS-tau-GFP labeled with GFP (J, J‴, arrows), Wrapper (J′, J‴) and BP102 (J″, J‴, asterisks).

Figure 2. Utility of wrapper-GAL4 in the study of CNS midline

(A, B) β-Gal staining of wrapper-GAL4/UAS-tau-lacZ stage 16 embryo show lacZ expression specifically in the MG at a low magnification (A) and high magnification (B). (C-E) Electron micrographs of embryonic stage 16 wild type midline displayed commissural axon tracts (Com) separated by MG (C). Also visible are the neuronal cell bodies in the CNS (asterisk). Higher magnification images shown with MG and their processes (arrowheads, D) and axons (Ax, E) surrounded by MG processes (arrowhead). (F, G) Electron micrographs of stage 16 wrapper-GAL4/UAS-tau-lacZ reveal MG cells and processes decorated by β-Gal reaction product crystals (F, G). Note the commissural axon tracts crossing the midline are surrounded by MG processes and are highlighted by β-Gal reaction product crystals (G). (H-L) Confocal images of stage 16 embryonic midline of wrapper-GAL4/UAS-wrapper-RNAi (I) and wrapper-GAL4/UAS-slit-RNAi (K) show a reduction in the levels of Wrapper (I, quantified in L) and Slit (K) compared to their wild type counterparts (H and J, respectively). Scale bars: C, F= 5μm; D, E= 0.2 μm; G= 2 μm. Anterior is to the left in A, B and to the top in H-K. a.u.= arbitrary units

Figure 3. wrapper-GAL4 and rescue of wrapper^-/- phenotypes

(A-D) Localization of Nrx IV (green), Wrapper (red) and BP102 (blue) in stage 16 embryos of wild type (A), nrx IV^-/- (B), wrapper^-/- (C) and wrapper^-/-;wrapper-GAL4/UAS-wrapper (D). Anterior is to the top in A-D. (E, F) Survival assay of embryonic to 1^st instar larvae (E) and 3^rd instar larvae to adults (F) in wild type, wrapper^-/- and wrapper^-/-;wrapper-GAL4/UAS-wrapper.

Figure 4. Contactin can substitute for Wrapper Function in MG to restore Nrx IV localization

(A) Comparison of domain structure of Drosophila Wrapper (WRAP) and Contactin (CONT). (B-E) Localization of Wrapper (blue), Nrx IV (green) and Cont (red) in wild type (B), wrapper^-/- (C) and wrapper^-/-;wrapper-GAL4/UAS-cont (D, E). Note the restoration of Nrx IV asymmetric localization (white arrowheads, D′, E′) upon Cont expression in MG (white arrowheads, D″ and E″). Higher magnification confocal image of MG in lateral view revealed localization of Cont in MG (white arrowhead, E″, E‴) and endogenous localization of Cont in channel glia (red arrowhead, E″, E‴). Anterior is to the top in B-E.