Glutamate receptor composition at Drosophila neuromuscular junctions depends on developmental stage and muscle identity
- Department of Neurobiology and Biophysics, University of Washington, United States
- Department of Neurobiology, University of Southern California, United States
Figures
Figure 1
Glutamate receptors at Drosophila neuromuscular junctions (NMJs).
(A) Phylogeny of 16 glutamate receptors in the Drosophila genome and their reported expression at the larval NMJ (based on Li et al., 2016). (B) Schematic of the larval Drosophila NMJ (top) and composition of glutamate receptors (bottom). Larval GluRs occur as two distinct tetrameric complexes, each with the same auxiliary subunit (Neto). (C) Example fluorescence images of larval and adult NMJs. Motor neurons are labeled by driving GFP with vGlut-GAL4 (green). Active zones are labeled with an antibody against Brp (Wagh et al., 2006) (magenta). Muscle is labeled with phalloidin (blue).
Figure 2 with 10 supplements
GluR subunit expression mapping in larval and adult muscles.
(A) Representative images showing GFP expression driven by a GAL4 reporter line (GluRIIC-GAL4) in larval and adult muscles. (B) Representative images showing antibody staining of GluRIIC at larval and adult neuromuscular junctions. (C) Summary table of GAL4 expression/antibody staining for all GluRs in larval and adult muscles. Antibodies and tagged proteins are combined in a single immunohistochemistry (IHC) column. Note that this initial screen focused on the coxa segment of the adult leg. See Figure 2—figure supplements 1–10 for representative images and Materials and methods for a complete list of reagents screened. (D) Representative images showing the GluR auxiliary subunit Neto-β tagged with GFP at larval and adult neuromuscular junctions.
Figure 2—figure supplement 1
Neuromuscular junction (NMJ) staining of larval body wall muscle.
Figure 2—figure supplement 2
Neuromuscular junction (NMJ) staining of adult abdomen muscle.
Figure 2—figure supplement 3
Neuromuscular junction (NMJ) staining of adult indirect flight muscle.
Figure 2—figure supplement 4
Neuromuscular junction (NMJ) staining of adult leg muscle (coxa).
Figure 2—figure supplement 5
GluRIIA expression in larval and adult muscles.
(A) Representative images showing GFP expression driven by a GAL4 reporter line (GluRIIA-GAL4) in larval and adult muscles. (B) Representative images showing antibody staining of GluRIIA::GFP protein (green) at larval and adult neuromuscular junctions (magenta).
Figure 2—figure supplement 6
GluRIIB expression in larval and adult muscles.
(A) Representative images showing GFP expression driven by a GAL4 reporter line (GluRIIB-GAL4) in larval and adult muscles. (B) Representative images showing antibody staining of GluRIIB protein (green) at larval and adult neuromuscular junctions (magenta).
Figure 2—figure supplement 7
GluRIID expression in larval and adult muscles.
(A) Representative images showing GFP expression driven by a GAL4 reporter line (GluRIID-GAL4, green) in larval and adult muscles. (B) Representative images showing antibody staining of GluRIID protein (green) at larval and adult neuromuscular junctions (magenta).
Figure 2—figure supplement 8
GluRIIE expression in larval and adult muscles.
(A) Representative images showing GFP expression driven by a GAL4 reporter line (GluRIIE-GAL4, green) in larval and adult muscles. (B) Representative images showing antibody staining of GluRIIE protein (green) at larval and adult neuromuscular junctions (magenta).
Figure 2—figure supplement 9
Representative confocal images showing GFP expression driven by a GAL4 reporter line (GluRIIC-GAL4, green) in six front legs from six flies.
Anterior views (top), posterior views (bottom).
Figure 2—figure supplement 10
Representative images showing GFP expression driven by a GAL4 reporter line (Clumsy-GAL4, green) in adult but not larval muscles.
Note that the Clumsy trojan GAL4 has a 3xP3-GFP marker left from its construction that gives faint glial GFP expression in the brain, vnc, and gut that can be seen in the larva. Larval expression was not detected when we screened the line with a UAS-mCherry reporter. Adult muscle expression is consistent with both fluorescent reporters.
Figure 3 with 4 supplements
Differential GluRIIB vs. GluRIIC subunit expression in adult muscle subtypes.
(A) Schematic of the fly’s front leg with annotations of three key muscle groups in the femur. (B) Femur with Neto-β::sfGFP (green) in neuromuscular junctions (NMJs; Brp, magenta) in all femur muscle types (phalloidin, blue). (C) 3D rendering of a confocal stack of femur muscles (phalloidin, blue) and GluRIIB GAL4>UAS-GFP (green). GFP is expressed specifically in tibia extensor muscle fibers. (D) Representative antibody staining images showing presence of GluRIIB in a tibia extensor muscle (left) and no detectable GluRIIB in an accessory tibia flexor muscle (right). (E) 3D rendering of a confocal stack of femur muscles (phalloidin, blue) and GluRIIC GAL4>UAS-GFP (green). GFP is expressed specifically in accessory tibia flexor muscle fibers. (F) Representative antibody staining images showing no detectable GluRIIC in a tibia extensor muscle (left) and presence of GluRIIC in an accessory tibia flexor muscle (right).
Figure 3—figure supplement 1
RNA-seq analysis of glutamate receptors in all adult muscles (Li et al., 2022).
Figure 3—figure supplement 2
RNA-seq analysis of glutamate receptors in all tissues of the adult leg (Li et al., 2022).
Figure 3—figure supplement 3
Table summarizing RNA-seq data of glutamate receptors in adult muscles (data from Li et al., 2022).
Figure 3—figure supplement 4
GluR expression in larval and adult motor neurons.
(A) Representative images showing expression of one example glutamate receptor subunit (GluRIB-GAL4, green) in larval and adult fly neurons. Motor neurons are visible as axons leaving the central nervous system. (B) Summary table of GAL4 expression in motor neurons.
Figure 4 with 1 supplement
GluClα is expressed extrasynaptically in adult fly muscles.
(A) Single-nucleus RNA-seq data show strong expression of GluClα in adult leg muscles. (B) A GAL4 reporter for GluClα labels muscles in adult legs but not larval muscles. Expression in the larva is mainly in the nervous system, as expected. (C) Representative images showing antibody staining of endogenously tagged GluClα::V5 in larval and adult muscles. Co-staining with an antibody against Brp reveals that GluClα does not co-localize with active zones. Labeling in larval muscle is likely from motor neurons (Li et al., 2021). (D) Labeling of GluClα protein with a V5 tag shows extrasynaptic expression along the edges of the muscle fibers, whereas active zones (magenta) are localized.
Figure 4—figure supplement 1
Validation of GluClα::V5 staining, showing positive controls (V5 staining in GluClα::V5 tagged flies) and negative controls (V5 staining in wild-type flies, no tag).
(A) Ventral nerve cord with nearby leg coxa muscle. Anti-V5 (green) localizes to the extrasynaptic muscle space (arrows) in the top fly (GluClα::V5) but not the bottom fly (wild type). (B) V5 staining positive and negative controls in the fly thorax. (C) V5 staining positive and negative controls in the central nervous system. Antibody against V5 (green), antibody against Brp (magenta), and muscle (phalloidin, blue).
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Mouse anti-Bruchpilot (nc82) antibody | Developmental Studies Hybridoma Bank | RRID:AB_2314866 | 1:50 |
| Antibody | Chicken anti-V5 | Fortis Life Sciences | A190-118A | 1:300 |
| Antibody | Rabbit anti-GFP | Thermo Fisher | A-11122 RRID:AB_221569 | 1:1000 |
| Antibody | Rat anti-FLAG | Novus Biologicals | NBP1-06712 | 1:200 |
| Antibody | Rabbit anti-GluRIIB | Perry et al., 2017 | n/a | 1:1000 |
| Antibody | Rabbit anti-GluRIIC | Goel and Dickman, 2018 | n/a | 1:100 |
| Antibody | Guinea pig anti-GluRIID | Perry et al., 2017 | n/a | 1:2000 |
| Antibody | Rabbit anti-GluRIIE | Dickman lab, this study | n/a | 1:2000 |
| Antibody | Rabbit anti-ALFA | Synaptic Systems N1581 | n/a | 1:500 |
| Antibody | Donkey anti-Rabbit Alexa Fluor 488 | Thermo Fisher | A-21206 RRID:AB_2535792 | 1:1000 |
| Antibody | Goat anti-Mouse Alexa Fluor 568 | Thermo Fisher | A-11004 RRID:AB_2534072 | 1:500 |
| Antibody | Goat anti-Rabbit Alexa Fluor 568 | Thermo Fisher | A-11011 RRID:AB_143157 | 1:500 |
| Antibody | Goat anti-Chicken Alexa Fluor 488 | Thermo Fisher | A11039 RRID:AB_2534096 | 1:250 |
| Antibody | Goat anti-Rat Alexa Fluor 568 | Thermo Fisher | A-11077 RRID:AB_2534121 | 1:500 |
| Antibody | Goat anti-Guinea Pig Alexa Fluor 568 | Thermo Fisher | A-11075 RRID:AB_2534119 | 1:250 |
| Antibody | Alexa Fluor Phalloidin Alexa Fluor 647 | Thermo Fisher | A22287 | 1:50 |
| Other | Tissue-Tek O.C.T. | EMS 62550-01 | n/a | Sample preparation |
| Other | Vectashield mounting medium | Vector Labs H-1000 | n/a | Sample preparation |
| Other | FocusClear | CelExplorer FC-101 | n/a | Sample preparation |
| Other | MountClear | CelExplorer MC-301 | n/a | Sample preparation |
| Software | FIJI | Schindelin et al., 2012 | RRID:SCR_002285 | Confocal image processing |
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Glutamate receptor composition at Drosophila neuromuscular junctions depends on developmental stage and muscle identity
eLife 15:RP111371.
https://doi.org/10.7554/eLife.111371.3
