Ephrin-B3 controls excitatory synapse density through cell-cell competition for EphBs
- Thomas Jefferson University, United States
- University of Pennsylvania, United States
- University of Delaware, United States
- Institute of Science and Technology Austria, Austria
- Howard Hughes Medical Institute, Stanford University, United States
Figures
Figure 1 with 1 supplement
Knockdown of ephrin-B3 does not alter synapse density in single-neuron microislands.
(a and b) Representative images of dendrites from single-neuron microislands transduced with control (pSuper) and eB3 shRNA viruses and immunostained at DIV21 for vGlut1 and PSD-95. Scale bar, 20 …
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Figure 1—source data 1
Knockdown of ephrin-B3 does not alter synapse density in single-neuron microislands.
- https://doi.org/10.7554/eLife.41563.003
Figure 1—figure supplement 1
Schematic of microisland experimental design.
(a) Single-neuron microislands provide a system devoid of potential competitive interactions. Blue dots represent endogenous PSD-95, green dots represent PSD-95-GFP (b) Two-neurons microislands …
Figure 2
PSD-95-GFP localizes to synapses and does not alter synapse density.
(a and b) Representative images of single-neuron microislands transfected with the indicated constructs and immunostained for GFP (for PSD-95-GFP), PSD-95 and vGlut1. Boxed regions are shown in …
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Figure 2—source data 1
PSD-95-GFP localizes to synapses and does not alter synapse density.
- https://doi.org/10.7554/eLife.41563.006
Figure 3
Synapse density is regulated by an ephrin-B3-dependent competitive mechanism.
(a) Representative images of control and competitive doublets. Neurons were immunostained for vGlut1, PSD-95 and GFP (for PSD-95-GFP) at DIV21. Arrows indicate untransfected (white arrow) and …
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Figure 3—source data 1
Synapse density is regulated by a competitive mechanism.
- https://doi.org/10.7554/eLife.41563.008
Figure 4 with 2 supplements
Effects of ephrin-B3 knockdown are rescued by unclustered EphB2 ectodomain treatment.
(a) DIV10 cortical neurons transfected with the indicated constructs along with GFP and treated with unclustered EphB2 ectodomain (EphB2-ecto). Arrowheads indicate colocalized PSD-95 (red) and …
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Figure 4—source data 1
Effects of ephrin-B3 knockdown are rescued byEphB2 ectodomain.
- https://doi.org/10.7554/eLife.41563.010
Figure 4—figure supplement 1
Validation of unclustered EphB2 ectodomain treatment method.
(a and b) Representative images of DIV10 cortical neurons transfected with GFP and either pSuper control vector (control) or eB3 shRNA and treated with 500 ng/ul (250 ng/well) EphB2 ectodomain …
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Figure 4—figure supplement 1—source data 1
Validation of EphB2 ectodomain treatment method.
- https://doi.org/10.7554/eLife.41563.012
Figure 4—figure supplement 2
Unclustered EphB2 ectodomain blocks competition in microislands.
(a and b) Representative images of control (a) and competitive (b) doublets treated with 500 ng/ml (250 ng/well) of unclustered EphB2 ectodomain. Scale bar, 20 μm. (c) Higher magnification images of …
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Figure 4—figure supplement 2—source data 1
EphB2 ectodomain blocks competition in microislands.
- https://doi.org/10.7554/eLife.41563.014
Figure 5 with 2 supplements
Efnb3 is expressed in CTIP2 +projection neurons.
(a) Representative cells within WT mouse cortex labeled by RNAscope ISH for CTIP2 mRNA (Bcl11b), SATB2 mRNA (Satb2), and eB3 mRNA (Efnb3). Scale bar, 3 μm. (b) Quantification of the number of …
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Figure 5—source data 1
Efnb3 is expressed in CTIP2 +projection neurons.
- https://doi.org/10.7554/eLife.41563.016
Figure 5—figure supplement 1
Schematic of recombination in eB3 MADM mice.
(a) Interchromosomal recombination in eB3 MADM mice generates fluorescently labeled cells of known eB3 genotype. Cre-mediated recombination in G2 phase followed by segregation of chromatids into …
Figure 5—figure supplement 2
Controls for RNAscope ISH.
(a) Representative three color RNAscope ISH images from WT and Efnb3-/- mouse cortex, showing reduced eB3 probe signal in CTIP2 + cells (CTIP2 + and CTIP2+/SATB2 + included) within Efnb3-/- cortex. …
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Figure 5—figure supplement 2—source data 1
Controls for RNAscope ISH.
- https://doi.org/10.7554/eLife.41563.020
Figure 6 with 1 supplement
Relative levels of ephrin-B3 control spine density in vivo.
(a) Representative large diameter (>1.6 μm) dendrites of subgranular pyramidal neurons in control and eB3 Nestin-spCre MADM mice. Scale bar, 3 μm. (b) Representative small diameter (<1.6 μm) …
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Figure 6—source data 1
Relative levels of ephrin-B3 control spine density in vivo.
- https://doi.org/10.7554/eLife.41563.022
Figure 6—figure supplement 1
Equal spine density in control MADM neurons regardless of fluorophore expressed.
(a) Representative large diameter (>1.6 μm) dendrites of subgranular pyramidal neurons in control Nestin-spCre MADM mice. Scale bar, 3 μm. (b) Quantification of spine density on large diameter …
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Figure 6—figure supplement 1—source data 1
Spine density WT MADM neurons.
- https://doi.org/10.7554/eLife.41563.024
Figure 7
Use of ephrin-B3 MADM mice to generate heterogenotypic cultures.
(a) Representative image of eB3 MADM (Emx1-Cre line) cortex containing sparse EGFP +and/or tdTomato +expressing cells. Scale bar, 100 μm. (b) Workflow diagram for generating eB3 MADM cultures. (c) …
Figure 8
Ephrin-B3 regulates local cell-cell differences in synapse density in MADM cultures.
(a and b) Representative image fields and corresponding masks used for analysis from control (a) and heterogenotypic (b) DIV11 MADM cultures. Scale bar, 20 μm. (c and d) Scatter plots in which each …
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Figure 8—source data 1
Ephrin-B3 regulates local cell-cell differences in synapse density in MADM cultures.
- https://doi.org/10.7554/eLife.41563.027
Figure 9
Control of synapse number by ephrin-B3 does not require activity.
(a and b) Representative image fields and corresponding masks used for analysis from control (a) and heterogenotypic (b) DIV11 MADM cultures treated with TTX (1 μm) from DIV3-11. Scale bar, 20 μm. (c…
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Figure 9—source data 1
Control of synapse number by ephrin-B3 does not require activity.
- https://doi.org/10.7554/eLife.41563.029
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus) | ephrin-B3 knockout (and WT littermates) | Yokoyama et al., 2001, Hruska et al., 2015, Antion et al., 2010. | RRID:MGI:3026744 | |
| Genetic reagent (M. musculus) | MADM-11 TG, eB3+/MADM-11 TG, eB3- (founder line) | This paper. | Progenitor: MADM-11 TG/TG (Hippenmeyer et al., 2010), RRID: IMSR_JAX:013749 | |
| Genetic reagent (M. musculus) | MADM-11 GT/GT; Emx1-Cre | Hippenmeyer et al., 2010, Beattie et al., 2017. | Progenitor: IMSR_JAX:005628 | |
| Genetic reagent (M. musculus) | MADM-11 GT/GT; Nestin-spCre | Hippenmeyer et al., 2010. | ||
| Strain, strain background (Rattus norvegicus domesticus) | Long Evans Rat | Charles River | Strain code: 006: RRID:RGD_2308852 | |
| Aantibody | Rabbit polyclonal anti-GFP | Thermo Fisher Scientific | A6455, RRID:AB_221570 | (1:2500) |
| Aantibody | Mouse monoclonal anti-PSD-95 | NeuroMab (clone 28/43) | Cat. # 75–028, RRID:AB_2292909 | (1:500) |
| Antibody | Guinea pig polyclonal anti-vGlut1 | Millipore | Cat. #AB5905, RRID:AB_2301751 | (1:5000) |
| Antibody | Rabbit polyclonal anti-RFP | Rockland antibodies | Cat. # 600-401-379, RRID:AB_2209751 | (1:500) |
| Antibody | Chicken polyclonal anti-GFP | Abcam | Cat. # ab13970, RRID:AB_300798 | (1:500) |
| Antibody | Rat monoclonal anti-CTIP2 | Abcam | Cat. # ab18465, RRID:AB_10015215 | (1:500) |
| Antibody | Mouse monoclonal anti-SATB2 | Abcam | Cat. # ab51502, RRID:AB_882455 | (1:500) |
| Antibody | Donkey polyclonal anti-rabbit Dylight 488 | Abcam | Cat. # ab96919, RRID:AB_10679362 | (1:500) |
| Antibody | Donkey polyclonal anti-chicken Dylight 488 | Jackson Immunoresearch | Cat. # ab96947, RRID:AB_10681017 | (1:500) |
| Antibody | Donkey polyclonal anti-rabbit Alexafluor 594 | Jackson Immunoresearch | Cat. # 711-585-152, RRID:AB_2340621 | (1:500) |
| Antibody | Donkey polyclonal anti-rat 647 | Jackson Immunoresearch | Cat. # 712-605-153, RRID:AB_2340694 | (1:500) |
| Antibody | Goat polyclonal anti-mouse ATTO 425 | Rockland antibodies | Cat. # 611-151-122, RRID:AB_10893217 | (1:250) |
| Antibody | Donkey polyclonal anti-guinea pig Dylight 649 | Jackson Immunoresearch | Cat. # 706-605-148, RRID:AB_2340476 | (1:500) |
| Recombinant DNA reagent | pFUGW-tdTomato_H1-eB3.2 (plasmid) | Hruska et al., 2015. | Progenitor: pFUGW (RRID:Addgene_14883); pSuper. | |
| Recombinant DNA reagent | pFUGW-tdTomato_H1-pSuper (plasmid) | Hruska et al., 2015. | Progenitor: pFUGW (RRID:Addgene_14883), pSuper. | |
| Recombinant DNA reagent | pSuper (plasmid) | Hruska et al., 2015, McClelland et al., 2010. | ||
| Recombinant DNA reagent | eB3.2 shRNA (plasmid) | Hruska et al., 2015, McClelland et al., 2010. | pSuper | |
| Recombinant DNA reagent | pFUGW (plasmid) | Hruska et al., 2015, McClelland et al., 2010. | RRID:Addgene_14883 | |
| Recombinant DNA reagent | pLL3.7 (plasmid) | Addgene | RRID:Addgene_11795 | |
| Recombinant DNA reagent | ephrin-B3 shRNA lentivirus | Penn Vector Core (University of Pennsylvania) | Progenitor: pLL3.7 vector (RRID:Addgene_11795) | |
| Peptide, recombinant protein | Recombinant human EphB2 Fc chimera | R and D systems | Cat. # 5189-B2 | |
| Peptide, recombinant protein | Recombinant Fc control fragment | R and D systems | 110-HG-100 | |
| Commercial assay or kit | RNAscope fluorescent multiplex kit | Advanced Cell Diagnostics | 320850 | |
| Commercial assay or kit | RNAscope probe- Mm-Bcl11b (CTIP2) | Advanced Cell Diagnostics | Cat. # 413271-C3 | |
| Commercial assay or kit | RNAscope probe- Mm-Satb2-C2 | Advanced Cell Diagnostics | Cat. # 413261-C2 | |
| Commercial assay or kit | RNAscope probe- Mm-Efnb3 | Advanced Cell Diagnostics | Cat. # 526771 | |
| Chemical compound, drug | Tetrodotoxin (TTX) | Tocris | Cat. #1078 | |
| Software, algorithm | NIH ImageJ | McClelland et al., 2009, Hruska et al., 2015. | RRID:SCR_003070 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.41563.030
