Meigo governs dendrite targeting specificity by modulating Ephrin level and N-glycosylation

Journal name:
Nature Neuroscience
Volume:
16,
Pages:
683–691
Year published:
DOI:
doi:10.1038/nn.3389
Received
Accepted
Published online

Abstract

Neural circuit assembly requires precise dendrite and axon targeting. We identified an evolutionarily conserved endoplasmic reticulum (ER) protein, Meigo, from a mosaic genetic screen in Drosophila melanogaster. Meigo was cell-autonomously required in olfactory receptor neurons and projection neurons to target their axons and dendrites to the lateral antennal lobe and to refine projection neuron dendrites into individual glomeruli. Loss of Meigo induced an unfolded protein response and reduced the amount of neuronal cell surface proteins, including Ephrin. Ephrin overexpression specifically suppressed the projection neuron dendrite refinement defect present in meigo mutant flies, and ephrin knockdown caused a similar projection neuron dendrite refinement defect. Meigo positively regulated the level of Ephrin N-glycosylation, which was required for its optimal function in vivo. Thus, Meigo, an ER-resident protein, governs neuronal targeting specificity by regulating ER folding capacity and protein N-glycosylation. Furthermore, Ephrin appears to be an important substrate that mediates Meigo's function in refinement of glomerular targeting.

At a glance

Figures

  1. meigo1 projection neuron dendrites are defective in mediolateral targeting and glomerular refinement.
    Figure 1: meigo1 projection neuron dendrites are defective in mediolateral targeting and glomerular refinement.

    (a) Schematic of the Drosophila olfactory system. ORNs (red) and projection neurons (PNs, green) target their axons and dendrites to genetically pre-specified target glomeruli (yellow) to generate one-to-one neural connections in the antennal lobe. Arrows indicate the flow of olfactory information. (b) Schematic of the MARCM projection neuron neuroblast clones of anterodorsal lineage labeled by GH146-Gal4. The target glomeruli of anterior surface of the antennal lobe are colored in green. The name of each glomerulus is also indicated. (c) Projection neurons derived from wild-type (WT) or meigo1 anterodorsal neuroblast MARCM clones were labeled by GH146-Gal4, Mz19-Gal4 or NP5103-Gal4. Dendrites of meigo1 projection neurons accumulated on the medial side of the antennal lobe. Yellow dotted lines indicate the target glomerulus of wild-type projection neurons. The white arrows indicate the spillover of dendrites. Green indicates mCD8-GFP–labeled projection neurons and magenta represents the presynaptic marker Brp. The white dotted lines indicate the midline. Scale bars represent 20 μm. (d) Quantification of the relative intensity of dendrite fluorescence of anterodorsal neuroblast clones (labeled by GH146-Gal4) along the mediolateral or dorsoventral axis of the antennal lobe. A significant medial shift of the dendrites was revealed, but no significant difference was observed dorsoventrally (wild type (n = 8) versus meigo1 (n = 10 individual MARCM clones): mediolateral, **P < 0.025; dorsoventral, not significant (n.s., P > 0.05)). Error bars indicate s.e.m. (e) Dendrite targeting of single-cell MARCM clones of the DL1 class of wild-type or meigo1 projection neurons. The yellow dotted lines indicate the DL1 glomerulus. Three examples of meigo1 projection neuron single-cell clones are shown. Normal: dendrites properly targeted and were restricted to the DL1 glomerulus. Mild: dendrites targeted around the DL1 glomerulus with spillover to neighboring (mostly medial) glomeruli. Severe: most dendrite branches mistargeted medial to DL1. Data are presented as in c.

  2. Targeting defects of meigo1 ORN axons.
    Figure 2: Targeting defects of meigo1 ORN axons.

    (a) Wild-type and meigo1 MARCM ORN clones induced by ey-FLP and visualized by pebbled-Gal4 or Or88a-Gal4. ORN axons were also misdirected medially in meigo1 clones. Green indicates mCD8-GFP–labeled ORNs. The yellow dotted lines indicate the target glomeruli of wild-type ORNs. Magenta represents the presynaptic marker Brp. The white dotted lines indicate the midline. Scale bars represent 20 μm. (b) Quantification of the relative intensity of axon fluorescence of MARCM clones (labeled by pebbled-Gal4) along the mediolateral or dorsoventral axis of the antennal lobe. A significant medial shift of the axons was revealed, but no significant difference was observed dorsoventrally (wild type (n = 6) versus meigo1 (n = 17): mediolateral, ***P < 0.001; dorsoventral, not significant (n.s., P > 0.05)). Error bars indicate s.e.m.

  3. Meigo is an ER resident protein that belongs to a family of NSTs.
    Figure 3: Meigo is an ER resident protein that belongs to a family of NSTs.

    (a) Predicted structure of the Meigo protein. A missense mutation in the meigo1 mutant (red star) is found in Cys89, which is located in the third transmembrane region. (b) Immunohistochemistry of an S2 cell with antibodies to Meigo (green), KDEL (red, an ER marker) and dGLG1 (also known as 120 kDa antibody; blue), which marks the Golgi. Endogenous Meigo was abundantly localized in the cytosol, in a pattern resembling internal membranous structures, and partially colocalized with the ER. Scale bars represent 5 μm. (c) Immunoblots of fractions from a membrane density gradient revealed that a significant portion of Meigo was present in fractions enriched with the ER membrane. The top and bottom of the gradient are indicated below the panels. The full-length blots are presented in Supplementary Figure 7. (d) A phylogenetic tree of Drosophila genes predicted to have NST activity. The subcellular localization is shown in parentheses. (e) Overexpression of the other NSTs, slalom, Efr or frc, did not suppress the dendrite mistargeting of meigo1 single-cell clones, suggesting that these NSTs cannot substitute for Meigo in projection neuron dendrite targeting. Green indicates projection neuron MARCM clones and magenta indicates Brp staining. Scale bars represent 20 μm.

  4. Meigo mediates ER homeostasis in projection neurons.
    Figure 4: Meigo mediates ER homeostasis in projection neurons.

    (a) Western blot analysis detected the unconventional splicing of xbp1-EGFP that was induced by ER stress in meigo-knockdown S2 cells. cat dsRNA was used as a negative control. Antibody to Meigo detects a 32-kDa band on a western blot, which corresponds to the predicted molecular weight of Meigo. Antibody to α-tubulin loading controls are shown below. The full-length blots are presented in Supplementary Figure 7. (b) Measurement of mRNA levels of unfolded protein response target genes (hsc3, pdi, herp, edem1 and edem2) by quantitative reverse transcription (RT)-PCR of S2 cell lysates treated with cat dsRNA or meigo dsRNA. Meigo depletion induced upregulation of UPR target gene expression (n = 4; *P < 0.05 for hsc3, pdi and herp). Error bars indicate s.e.m. (c) MARCM expression of xbp1-EGFP in wild-type or meigo1 neuroblast clone of lateral lineage. The Xbp1-EGFP accumulated in the nuclei of meigo1, but not wild-type, projection neurons. Red indicates mCD8-RFP–labeled projection neurons, and blue represents the presynaptic marker Brp. Scale bars represent 20 μm. (d) Hsc3 staining (red) in wild-type or meigo1 neuroblast clones. The enhanced Hsc3 signals in the medially shifted dendrites were observed only in meigo1 neuroblast clones. Green marks mCD8-GFP–labeled projection neurons. Blue represents the presynaptic marker Brp. Scale bars represent 20 μm. (e) Quantification of the severity of the phenotype of meigo1 single-cell clones overexpressing molecules that induce ER stress. Expression of Hsc3K97S, Hsc3D231S or ninaEG69D in wild-type (+) single-cell clones had either a weak effect or no effect; however, expression in meigo1 (1) single-cell clones enhanced the targeting defect, suggesting that mutation of meigo enhances sensitivity to the ER stress.

  5. ephrin regulates projection neuron dendrite targeting and genetically interacts with meigo.
    Figure 5: ephrin regulates projection neuron dendrite targeting and genetically interacts with meigo.

    (a) Quantification of the phenotype of meigo1 single-cell clones overexpressing various cell-surface molecules. Expression of full-length ephrin or myc-tagged ephrin specifically suppressed dendrite mistargeting in meigo1 single-cell clones. (b) The protein level of transfected ephrin-myc in S2 cells was verified by western blot (n = 5). The α-tubulin loading controls are shown below. The quantification of Ephrin:myc normalized with α-tubulin revealed a significant decrease in meigo-depleted cells compared with control (**P < 0.025). Error bars indicate s.e.m. The full-length blots are presented in Supplementary Figure 7. (c) Immunohistochemistry of an S2 cell with antibodies to Ephrin (green), Meigo (red) and Pdi (blue). The ratio of subcellular localization of Ephrin-myc under the condition of knockdown or overexpression is indicated in right. Scale bars represent 5 μm. (d) The dendrite innervation pattern of wild-type or ephrin shRNA–expressing anterodorsal MARCM clones labeled by GH146-Gal4 or NP5103-Gal4. A neuroblast clone expressing ephrin shRNA exhibited spillover from glomeruli with no discernible direction (left). NP5103-Gal4–labeled neruroblast clones (middle) and GH146-Gal4–labeled single cell clone (right) also showed the spillover from appropriate glomerulus, indicated by white arrows. Green marks mCD8-GFP–labeled projection neurons. Magenta represents the presynaptic marker Brp. Scale bars represent 20 μm.

  6. Meigo positively regulates the N-glycosylation of Ephrin that is important for its function in vivo.
    Figure 6: Meigo positively regulates the N-glycosylation of Ephrin that is important for its function in vivo.

    (a) Structure of the Ephrin-myc protein. The three transmembrane domains (gray boxes) and ephrin domain (orange box) are predicted. The four N-glycosylation sites are Asn222, Asn265, Asn274 and Asn365, which exist around the ephrin domain (orange branches). In EphrinNQ-myc, all of the N-glycosylation sites were mutated from Asn to Gln. The C terminus is fused with five copies of myc tags (pink bar). (b) N-glycosidase (PNGaseF) sensitivity of ephrin-myc or ephrinNQ-myc expressed in S2 cells. The PNGaseF treatment to the ephrin-myc resulted in a band shift to the comparable molecular weight of ephrinNQ-myc, indicating that ephrin-myc is N-glycosylated. (c) Immunoblot analysis showing the N-glycosylation state of ephrin-myc in either meigo knockdown or overexpression. The highest band (N-Gly4) represents all of the four predicted sites being N-glycosylated, the second highest band (N-Gly3) indicates that three sites are N-glycosylated, and so on. Middle, immunoblot with antibody to Meigo, which shows effective knockdown and overexpression of meigo. The α-tubulin loading controls are shown below. Full-length blots for the data shown in b and c are presented in Supplementary Figure 7. (d) Quantification of the relative ratio of N-Gly4, N-Gly3, N-Gly2, N-Gly1 and N-Gly0. Meigo positively regulated the ratio of N-Gly4 (n = 5 independent experiments, **P < 0.01, ***P < 0.001). Error bars indicate s.e.m. (e) Quantification of the phenotype of meigo1 single-cell clones overexpressing ephrin-myc or ephrinNQ-myc. The suppression efficiency was apparently weaker in ephrinNQ-myc clones than in ephrin-myc clones.

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Affiliations

  1. Department of Genetics, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan.

    • Sayaka U Sekine,
    • Shuka Haraguchi,
    • Kinhong Chao,
    • Tomoko Kato,
    • Masayuki Miura &
    • Takahiro Chihara
  2. Howard Hughes Medical Institute and Department of Biology, Stanford University, Stanford, California, USA.

    • Liqun Luo
  3. Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Tokyo, Japan.

    • Masayuki Miura
  4. Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Tokyo, Japan.

    • Takahiro Chihara

Contributions

S.U.S. performed most of the experiments and analyzed the data. S.H., K.C. and T.K. assisted in some experiments. T.C. supervised the project. S.U.S. and T.C. wrote the paper with feedback from L.L. and M.M.

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The authors declare no competing financial interests.

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