Drosophila Tet Is Expressed in Midline Glia and Is Required for Proper Axonal Development

Abstract

Ten-Eleven Translocation (TET) proteins are important epigenetic regulators that play a key role in development and are frequently deregulated in cancer. Drosophila melanogaster has a single homologous Tet gene (dTet) that is highly expressed in the central nervous system during development. Here, we examined the expression pattern of dTet in the third instar larval CNS and discovered its presence in a specific set of glia cells: midline glia (MG). Moreover, dTet knockdown resulted in significant lethality, locomotor dysfunction, and alterations in axon patterning in the larval ventral nerve cord. Molecular analyses on dTet knockdown larvae showed a downregulation in genes involved in axon guidance and reduced expression of the axon guidance cue Slit. Our findings point toward a potential role for dTet in midline glial function, specifically the regulation of axon patterning during neurodevelopment.

Keywords: Drosophila; Tet; axon guidance cue; axon guidance defect; midline glia; neurodevelopment.

FIGURE 1

dTet is expressed in neurons and a subset of glia in the third instar larval CNS. (A) Left, maximum intensity projection at low magnification showing overlap between dTet-GFP in Elav-positive neurons. Scale bar, 50 μm. Right, high magnification micrograph of neurons in central brain co-expressing Elav and dTet-GFP. Arrowhead indicating midline glia pattern. Scale bar, 20 μm. (B) Left, maximum intensity projection at low magnification showing expression of dTet-GFP and Repo-positive glial cells. Scale bar, 50 μm. Right, high magnification maximum intensity projection showing expression of dTet-GFP within glia in the optic lobe (arrow). Scale bar, 20 μm. CB, central brain, VNC, ventral nerve cord, OL, optic lobe.

FIGURE 2

dTet is present in midline glial cells in the ventral nerve cord of third instar larval CNS. (A) In wild-type controls, anti-Slit labels midline glial cells in the ventral nerve cord. In dTet-GFP flies, dTet, and Slit are co-expressed. Scale bar, 50 μm. (B) High magnification confocal micrograph showing the presence of dTet in midline glia. Scale bar, 10 μm.

FIGURE 3

dTet knockdown affects locomotor behavior and leads to reduced survival. (A) Left, survival assay on third instar larvae. dTet knockdown larvae show reduced survival rates in ubiquitous (Tubulin-Gal4) and midline glial (Slit-Gal4) knockdown (p < 0.0001, n = 3, 20 larvae per group). Right, crawling assay on third instar larvae. dTet knockdown larvae exhibit reduced locomotor performance in both ubiquitous and midline glia-specific groups as compared to controls (p < 0.0001, n = 50). Control is Tubulin-Gal4 > w1118. Mean is shown with SEM. (B) Maximum intensity projections of larval VNCs with axon commissures labeled with FasIII. Asterisks indicate defects in commissure crossing. Scale bar, 20 μm. Inset, 20 μm. (C) Quantification of axon defects in control and knockdown VNCs (n = 30) where 0, 1, and 2+ errors are indicated.

FIGURE 4

dTet knockdown is associated with downregulation of axon guidance genes and reduced expression of Slit protein. (A) qRT-PCR on candidate genes in dTet knockdown larvae showing transcript levels normalized to Rp49 in Tubulin-Gal4 > w1118. (n = 3, 15 larvae per group). Mean is shown with SEM. (B) Representative Western blot on protein extracts from larval brains. The 180-kDa band corresponding to the Slit protein is reduced in dTet knockdown brains compared to controls (p < 0.05, n = 3, 35 brains per group). (C) Representative images from dot blot assays on 6mA and 5hmrC abundance in third instar larval brains (n = 3). Methylene blue (MB) used as loading control; 600 ng for DNA and 1500 ng for RNA. C: Control (Tubulin-Gal4 > w1118), KD: dTet knockdown (Tubulin-Gal4 > dTet-RNAi). Graphs showing relative abundance of 6mA and 5hmrC marks in knockdown larvae normalized to loading control (p < 0.05 and p < 0.01, respectively, n = 3). Mean is shown with SEM.