Synapse maintenance and restoration in the retina by NGL2
- Washington University School of Medicine, United States
Figures
Figure 1 with 1 supplement
AAV-mediated knockout of Ngl2 in horizontal cells.
(A) Schematic illustrating AAV-mediated CRISPR/Cas9 strategy for Ngl2 knockout in horizontal cells. In AAV-sgNGL2-tdT, small guide RNAs targeting NGL2 (sgNGL2) were expressed from a Pol III U6 …
Figure 1—figure supplement 1
NGL2 localizes to tips of horizontal cell axons, not rod bipolar cell dendrites.
(A and B) Representative images of axon tips of horizontal cells infected with AAV-YFP (A, injection at P0, imaging at P30) or dendrites of rod bipolar cells infected with AAV-Grm6-YFP (B) in …
Figure 2 with 3 supplements
NGL2 regulates horizontal cell axon size and tip density cell autonomously.
(A–E) Representative images of horizontal cell axons labeled by AAV-YFP (YFP) or AAV-sgNGL2-tdT (sgNGL21 and sgNGL22) in wild-type (WT) and Cas9 mice. Overview images are maximum intensity …
Figure 2—figure supplement 1
Nearly all horizontal cell axon tips are synaptic.
(A and B) Representative images of axon tips of horizontal cells infected with AAV-YFP in a wild-type retina (A) and with AAV-sgNGL2-tdT in a Cas9 retina (B) stained for the presynaptic ribbon …
Figure 2—figure supplement 2
Stable changes in horizontal cell axon size and axon tip density after NGL2 removal.
(A) Schematic illustrating experimental timeline. AAVs were intravitreally injected into newborn (P0) mice and retinas collected on P30, P60, and P90. (B and C) Representative images of horizontal …
Figure 2—figure supplement 3
Effects of NGL2 removal on horizontal cell axon targeting.
(A–F) Representative images of horizontal cell axons – rotated maximum intensity projections of confocal image stacks from retinal whole mounts – labeled by AAV-YFP (YFP, (A, C, F) or AAV-sgNGL2-tdT …
Figure 3 with 1 supplement
NGL2 restrains horizontal cell axon growth and maintains axon tips in young adult mice.
(A) Schematic of the experimental timeline. AAVs were injected into the vitreous chamber of P30 mice and retinas collected on P60. (B and C) Representative images of horizontal cell axons labeled by …
Figure 3—figure supplement 1
NGL2 restrains HC axon growth and maintains axon tips in adult mice.
(A) Schematic of the experimental timeline. AAVs were injected into the vitreous chamber of P30 mice and retinas collected at P90. (B and C) Representative images of axons of horizontal cells …
Figure 4 with 1 supplement
NGL2 restrains horizontal cell axon growth and maintains axon tips in mature adult mice.
(A) Schematic of the experimental timeline. AAVs were injected into the vitreous chamber of P150 mice and retinas collected on P180. (B and C) Representative images of horizontal cell axons labeled …
Figure 4—figure supplement 1
Small guide RNAs targeting Ngl2 do not affect horizontal cell axons in mature adult wild-type mice.
(A) Representative image of the axon of a horizontal cell infected with AAV-sgNGL2-tdT (sgNGL2) in a wild-type retina. Overview images are maximum intensity projections of the complete axons; insets …
Figure 5 with 1 supplement
AAV-mediated NGL2 expression normalizes axon growth and enhances axon tip formation of horizontal cells in developing Ngl2-/- mice.
(A) Schematic of the experimental timeline. AAVs were injected into the vitreous chamber of P0 mice and retinas collected at P30. (B–E) Representative images of axons of horizontal cells infected …
Figure 5—figure supplement 1
AAV-mediated NGL2 expression in Ngl2-/- and wild-type P30 retinas.
(A and B) Higher magnification excerpts of overview images in Figure 5 (C) and (E). We adjusted the brightness of the images in this figure supplement to reveal wild-type NGL2 expression in (B) and …
Figure 6 with 1 supplement
AAV-mediated NGL2 expression shrinks axon arbors and restores axon tips of horizontal cells in adult Ngl2-/- mice.
(A) Schematic of the experimental timeline. AAVs were injected into the vitreous chamber of P30 mice and retinas collected at P60. (B–E) Representative images of axons of horizontal cells infected …
Figure 6—figure supplement 1
AAV-mediated NGL2 expression in Ngl2-/- and wild-type P60 retinas.
(A and B) Higher magnification excerpts of overview images in Figure 6 (C) and (E). We adjusted the brightness of the images in this figure supplement to reveal wild-type NGL2 expression in (B) and …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Mouse strain | Wild-type (C57Bl6/J) | Jackson Laboratory | RRID:IMSR_JAX000664 | |
| Mouse strain | Cas9 | Jackson Laboratory | RRID:IMSR_JAX:024858 | |
| Mouse strain | Ngl2-/- | (Soto et al., 2013) | ||
| Plasmid | pX330-U6-Chimeric_BB-CBh-hSpCas9 | Addgene | Plasmid # 42230 | |
| Plasmid | pmaxGFP | Lonza | Catalog # VDC-1040 | |
| Virus | AAV-YFP | This paper | ||
| Virus | AAV-Grm6-YFP | (Johnson et al., 2017) | ||
| Virus | AAV-sgNGL21-tdT | This paper | ||
| Virus | AAV-sgNGL22-tdT | This paper | ||
| Virus | AAV-NGL2 | This paper | ||
| Antibody | anti-NGL2 (mouse) | NeuroMab | RRID:AB_2137614 | 1:100 dilution |
| Antibody | anti-Bassoon (mouse) | Enzo | RRID:AB_2313990 | 1:500 dilution |
| Antibody | anti-DsRed (rabbit) | Clontech Laboratories | RRID:AB_10013483 | 1:1000 dilution |
| Antibody | anti-GFP (chicken) | ThermoFisher | RRID:AB_2534023 | 1:500 dilution |
| Antibody | anti-GFP (rabbit) | ThermoFisher | RRID:AB_221569 | 1:1000 dilution |
| Antibody | anti-chicken IgY Alexa 488 | ThermoFisher | RRID:AB_2534096 | 1:1000 dilution |
| Antibody | anti-rabbit IgG Alexa 488 | ThermoFisher | RRID:AB_2536097 | 1:1000 dilution |
| Antibody | anti-rabbit IgG Alexa 568 | ThermoFisher | RRID:AB_143011 | 1:1000 dilution |
| Antibody | anti-mouse IgG Alexa 568 | ThermoFisher | RRID:AB_2534072 | 1:1000 dilution |
| Antibody | anti-mouse IgG Alexa 633 | ThermoFisher | RRID:AB_2535718 | 1:1000 dilution |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.30388.016
