EphB1T697G, EphB2T699A, and EphB3T706A single mutants were generated individually by homologous recombination in mouse embryonic stem (ES) cells. Mice harboring each of the KI mutations were intercrossed to obtain the triple homozygous AS-EphB TKI mice.
To generate the targeting constructs, the 5’ and 3’ arms was PCR amplified from J1 ES cell DNA using primers listed in Supplementary Table 1
and subcloned into a modified pKSNeoDTA vector (originally constructed in the lab of Philippe Soriano) containing a loxP-Neo-loxP cassette for positive selection and a diptheria toxin A negative selection cassette (DTA). The AS mutations were introduced by site-directed mutagenesis. The targeting constructs were confirmed by sequencing. Linearized targeting construct was electroporated into J1 ES cells, which were subsequently selected with G418. Correct targeting of ES cells was initially screened by PCR and then confirmed by Southern analysis and direct sequencing of PCR products amplified from the mutated alleles. The positive clones were karyotyped and the Neo cassette was removed by electroporating targeted ES cells with a Cre expression plasmid. ES clones were microinjected into C57BL/6 blastocysts to generate chimeric mice. Male chimeric animals were mated to C57BL/6 wild-type females for germline transmission of the targeted allele.
The ES cell targeting efficiencies were as follows: EphB1 KI (17/192 = 9%), EphB2 KI (3/186 = 2%), EphB3 KI (12/96 = 13%).
Mice were maintained as homozygotes in a mixed 129/C57BL/6 background. Unless noted, wild-type (wild-type) mice were F1 offspring of a C57BL/6 x129sv cross. Animals were housed under a 12-hour light/dark cycle. No more than five animals were housed in each cage. Mice and embryos were chosen at random, regardless of sex, for treatment condition.
All experiments with mice were approved by the Animal Care and Use Committee of Harvard Medical School. Embryonic day 0 (E0) was defined as midnight preceding the morning a vaginal plug was found.
HEK 293 cell culture and transfection
HEK 293 cells were maintained in DMEM supplemented with 10% fetal bovine serum (Gibco), 2mM glutamine (Gibco), and penicillin/streptomycin (100 U/mL and 100 µg/mL, respectively; Gibco). HEK 293 cells were transfected using the calcium phosphate method as previously described15
Antibodies and Western blotting
The following antibodies were purchased commercially: pan-phospho-tyrosine pY99 mouse monoclonal (Santa Cruz); EphB1 (H-80) and EphB3 (H-85) rabbit polyclonal (Santa Cruz); β-actin mouse monoclonal (Abcam); PSD-95 mouse monoclonal (Pierce); synapsin rabbit polyclonal (Millipore). Anti-neurofilament (2H3) mouse monoclonal antibody was obtained from the Developmental Studies Hybridoma Bank (from Thomas Jessell). Vav2, pan-phospho-EphB, and EphB2 rabbit polyclonal antibodies were generated in the Greenberg lab and described previously15
. The phospho-specificity of the pan-phospho-EphB antibody was validated in HEK 293 cells for EphB1, EphB2, and EphB3 individually.
All Western blots were imaged and quantified using the Odyssey Infrared Imaging System (Licor) using fluorescently labeled secondary antibodies (Rockland Immunochemicals).
Protein binding assays
Full length human Grb2 and rat Pick1 were PCR amplified from Plasmids #26085 and #31613 (Addgene), respectively. Grb2 and Pick1 were fused to GST at their N-termini by cloning them into a pGEX vector (Pharmacia), expressed in E. coli, and affinity purified on glutathione sepharose beads. GST, used as the negative control, was expressed from the empty pGEX vector. EphB1AS protein was expressed in HEK 293 cells by transient transfection and treated with DMSO or 1 µM 1-NA-PP1 for 16 hrs. Cells were lysed in lysis buffer (30 mM Hepes pH 7.7, 100 mM KCl, 1 mM MgCl2, 2 mM DTT, 2 mM sodium orthovandadate, 1% Triton X-100, protease inhibitor (Roche, 04693159001) and phosphatase inhibitor cocktails (Sigma, P5726, P0044)). The crude lysates were centrifuged at 40,000 rpm for 20 minutes at 4 degree C to generate the high-speed supernatants (HSS). HSS was incubated with glutathione beads coated with approximately GST, GST-Grb2 or GST-Pick1 with rotation at 4 degree C for 2 hours. The beads were washed 3 times with lysis buffer and analyzed by SDS-PAGE and Western blotting.
1-NA-PP1 was synthesized as described previously27
and dissolved in DMSO. 3-MB-PP1 was synthesized using a similar procedure and dissolved in DMSO. Dose response curves using 1-NA-PP1 and 3-MB-PP1 were calculated on GraphPad Prism using the least-squares method. The vehicle dose was calculated as two orders of magnitude below the lowest dose (=0.05 nM). For wild-type EphBs, 100% inhibition was defined at 1 mM.
Neuronal cell culture
Cortical and hippocampal neurons were prepared from E15-E17 mouse embryos as previously described15
. Cultured neurons were maintained in Neurobasal Medium (Invitrogen) supplemented with 1× B27 (Invitrogen), penicillin/streptomycin (100 U/mL and 100 µg/mL, respectively) and 2mM glutamine. For biochemistry, neurons were seeded at a density of 2×10^6
neurons/well of a 6-well plate coated with polyornithine (Sigma). For electrophysiology and imaging, neurons were seeded at a density of 7.5×10^4
neurons/well on a glial monolayer on glass coverslips coated with polyornithine and laminin (Invitrogen).
Hippocampal organotypic slices were prepared in ice-cold dissection media (1 mM CaCl2, 5 mM MgCl2, 10mM D-glucose, 4 mM KCl, 26 mM NaHCO3, 218 mM sucrose, 1.3 mM sodium phosphate, and 30 mM HEPES, pH 7.4). Brains were isolated from P5-7 pups, and hippocampi were excised and chopped into 400 µm sections. Slices were cultured on Millicell cell culture inserts (Millipore) in media containing 20% horse serum, 1 mM L-glutamine, 0.0012% ascorbic acid, 1 µg/mL insulin, 1 mM CaCl2, 2 mM MgCl2, 2.3 mg/mL glucose, 0.44 mg/mL NaHCO3, 7.16 mg/mL HEPES in MEM.
For ephrin stimulations in dissociated cultured neurons and retinal explants, mouse ephrin-B1-Fc or ephrin-B2-Fc (R&D Systems) was pre-clustered for 50 minutes with goat anti-human IgG Fc (Jackson Immunoresearch) at room temperature in PBS at a molar ratio of 1:1 prior to stimulation. Pre-clustered ephrin-B1-Fc or ephrin-B2-Fc was added to the appropriate medium at a final concentration of 2.5 µg/mL. As a control, clustered human Fc in media was applied to neurons where specified.
Cell lysis and immunoprecipitation
Cultured cells were collected and homogenized in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% Sodium Deoxycholate, 0.1% SDS, 10 mM NaF, complete protease inhibitor cocktail tablet (Roche), 1 mM sodium orthovanadate, and Phosphatase Inhibitor cocktails 1 and 2 (1×; Sigma). After clearing lysates, supernatants were incubated with the appropriate antibody for 1 hour at 4°C, followed by addition of Protein-A Fastflow agarose beads (Sigma) for 1 hour. Beads were washed in lysis buffer or PBS three times and eluted in 2× SDS sample buffer followed by boiling.
Labeling of surface proteins was performed using the Pierce Cell Surface Protein Isolation Kit (Thermo Scientific). After chronic treatment with vehicle or 1 µM 1-NA-PP1, cultured cortical neurons were incubated with EZ-link biotin for 30 minutes at room temperature, washed with PBS, and lysed in RIPA buffer. Lysates were immunoprecipitated with an anti-EphB2 or control antibody, and probed with an anti-EphB2 antibody or fluorescently labeled streptavidin (Invitrogen).
Ventrotemporal (VT) segments of retina were microdissected from E14.5 mouse embryos and cultured as previously described 51
. Embryos were removed from the uterus and decapitated, and heads were placed in ice-cold DMEM/F12 (Gibco). VT sections of the retina were excised and placed on glass coverslips coated with polyornithine and laminin. Explants were maintained in serum free medium (10 mg/mL BSA (Sigma), 1% ITS supplement (Sigma), Pen/Strep (20 U/mL and 20 µg/mL, respectively, Gibco) in DMEM/F12) supplemented with 0.2% methyl cellulose (Sigma) to increase media viscosity and minimize explant movement. All experiments were conducted 18–24 hour after initial plating.
Immunocytochemistry and growth cone collapse assay
For experiments with inhibitor treatment, explants were pre-incubated with vehicle or PP1 analogs for 1 hour, followed by a 30-minute ephrin-B2 stimulation, with variations as described in the text. Following stimulation, retinal explants were fixed for 20 minutes at 25°C with 4% paraformaldehyde (PFA)/2% sucrose in PBS. Explants were then blocked in 10% goat serum, 0.2% Tween-20 in PBS for 1 hour, followed by incubation with an anti-neurofilament antibody or anti-phospho-EphB antibody in 50% blocking solution overnight. After PBS washes, explants were incubated in Alexa Fluor-conjugated secondary antibodies (Invitrogen) and Alexa Fluor 488-conjugated phalloidin (Invitrogen). Explants on coverslips were mounted on glass slides using Fluoromount-G (Southern Biotech). Neurons were imaged using a laser scanning Zeiss Pascal microscope using a 40× objective with sequential acquisition settings at 1024 × 1024 pixel resolution. All imaging and image analysis were performed blind to the genotype and treatment condition of the samples. At least 10 growth cones were analyzed per explant.
In vivo 1-NA-PP1 delivery
Pregnant wild-type or AS-EphB TKI mice were injected subcutaneously twice daily with 80mg/kg 1-NA-PP1 dissolved in 10%DMSO, 20% Cremaphor-EL, 70% saline from E13.5 to E16.5 for optic tract experiments or from E12.5-E19 for cortical tract experiments. All experiments included data from at least two separate litters of embryos per condition. Animals used for in vivo 1-NA-PP1 treatment had no prior exposure to 1-NA-PP1 or other drugs.
DiI labeling was performed as previously described52
. At E16.5, embryo heads were fixed in 4%PFA /2% sucrose in PBS overnight and then washed with PBS. The lens and retina were removed from the left eye and a small crystal of DiI (Invitrogen) was placed in the optic disc. The retina was then replaced securely and the heads were stored in PBS+0.1% azide at room temperature for 12 days. After labeling, brains were removed and fluorescent optic tracts were imaged on a Leica MZ16F fluorescent stereomicroscope. Images were captured using Spot Advanced software. Labeling was quantified using Metamorph software by drawing rectangular regions of interest around the ipsilateral and contralateral tracts, subtracting background, and calculating the ipsilateral index based on integrated intensity of fluorescence:
- ipsilateral index = ipsilateral / (ipsilateral + contralateral).
To compare wild-type and AS-EphB TKI responses with respect to 1-NA-PP1 treatment, each genotype was normalized to its untreated condition, producing a normalized ipsilateral index.
Analysis of corpus callosum phenotypes
E19.0 embryos were fixed in 4% PFA/2% sucrose in PBS for 2 days, then stored in PBS + 0.02% sodium azide at 4°C. Brains were removed and vibratome sectioned to 70 µm. Sections were blocked in 5% normal donkey serum, 1% BSA, 0.2% glycine, 0.2% lysine with 0.3% TritonX-100 in PBS at room temperature for 1 hour. To stain axon tracts, sections were incubated with rat anti-L1-CAM (Millipore, MAB5272MI) at a 1:200 dilution in blocking solution at 4°C overnight. Sections were washed three times with PBS and incubated with Cy3-conjugated donkey anti-rat IgG (Jackson) for 1.5 hours. Sections were washed 7–8 times with PBS and mounted with Aqua-mount (Lerner laboratories). Sections were imaged on an Olympus Bx51 epifluorescent microscope at 4× magnification. Corpus callosum partial agenesis was scored as the apparent failure of corpus callosal axons to cross the midline in a specific region along the rostral-caudal brain axis, and was evident by a gap in the dorsal midline region.
Total RNA was isolated from mouse 7 DIV cortical cultures using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. Isolated RNA was treated with DNAseI Amplification Grade (Invitrogen) and a cDNA library was synthesized by cDNA High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The cDNA was the source of input for quantitative PCR, using a Step One Plus Real-Time PCR Instrument and SYBR Green reagents (Applied Biosystems). The relative expression plot was generated using concentration values that were normalized to corresponding actin concentrations. The following qPCR primer pairs were used:
- EphB1-F- ACTGCAGAGTTGGGATGGAC
- EphB1-R- CATCATAGCCACTGACTTCTTCC
- EphB2-F- TTCATGGAGAACGGATCTCTG
- EphB2-R- GACTGTGAACTGCCCATCG
- EphB3-F- CCCTGGACTCCTTTCTACGG
- EphB3-R- GCAATGCCTCGTAACATGC
Analysis of dendritic spines
For dissociated neuron experiments, cortical neurons were cultured as described above and grown on a monolayer of astrocytes on glass coverslips. Neurons were treated with vehicle or PP1 analogs at 10 DIV and media was changed entirely every 3–4 days (using neuronally pre-conditioned media). Neurons were transfected with GFP at 10 DIV using Lipofectamine 2000 and fixed with 4%PFA/2%sucrose in PBS at 21 DIV. Cells on coverslips were stained for GFP and mounted on slides using Fluoromount-G.
Neurons were imaged on a Zeiss Pascal confocal microscope, using a 63× objective, and maximal z-projections were analyzed using Metamorph software. Multiple sections of dendrite totaling > 50 µM were counted for each neuron.
For experiments in slice, hippocampal slices were treated with vehicle or PP1 derivatives at 2 DIV and media/drug were fully replaced every 2–3 days. At 2–3 DIV, plasmids encoding GFP (or GFP + EphB shRNAs) was biolistically transfected using a Helios gene gun. DNA bullets were prepared from 1.6 µm gold microcarrier particles (Biorad). After 8–9 DIV, slices were fixed in 3.2% PFA/5% sucrose in PBS for 1 hour and stained with chicken anti-GFP (Aves Labs) and rabbit anti-NeuN (Millipore) antibodies to visualize the structure of hippocampal fields. Basal and apical dendrites were analyzed separately and sections of dendrite totaling > 50 µM were counted for each neuron.
For experiments in dissociated neurons, whole-cell voltage clamp recordings were obtained using an Axopatch 200B amplifier at 25°C. During recordings, neurons were perfused with artificial cerebrospinal fluid containing 127 mM NaCl, 25 mM NaHCO3, 1.25 mM Na2HPO4, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 25 mM glucose, and saturated with 95% O2, 5% CO2. Vehicle or 1-NA-PP1 treatment was initiated at 3 DIV and continued throughout recordings. The internal solution used in all eletrophysiological experiments contained 120 mM cesium methane sulfonate, 10 mM HEPES, 4 mM MgCl2, 4 mM Na2ATP, 0.4 mM Na2GTP, 10 mM sodium phosphocreatine and 1 mM EGTA. Osmolarity and pH were adjusted to 310 mOsm and 7.3 with Millipore water and CsOH, respectively.
mEPSCs were isolated by exposing neurons to 0.5 µM tetrodotoxin, 50 µM picrotoxin, and 10 µM cyclothiazide (all from Tocris Bioscience). Cells with series resistance larger than 25 MΩ during the recordings were discarded. Data were analyzed in IgorPro (Wavemetrics) using custom-written macros. For each trace, the event threshold was set at 1.5 times the root-mean-square current. Currents were counted as events if they crossed the event threshold, had a rapid rise time (1.5 pA ms-1) and had an exponential decay (tau<50 ms for mEPSC).
As a control for inhibition of EphBs, concurrent plates of neurons were treated with inhibitor and at the time of recording were stimulated with ephrin-B1 for 30 minutes. Neurons were lysed in 1× SDS-sample buffer, run on western blot and probed with rabbit anti-phospho-Eph and mouse anti-β-actin (Abcam) antibodies.
For mEPSC experiments in organotypic hippocampal slices, whole-cell voltage clamp recordings were made from visually identified CA1 pyramidal neurons and the mEPSC amplitude and frequency measured. Slices were treated with vehicle or 1-NA-PP1 from 2 DIV until the time of recording.
To evaluate evoked synaptic transmission, the Schafer collaterals were depolarized with an extracellular stimulating electrode and the postsynaptic evoked EPSC (eEPSC) response measured from CA1 neurons53
. In these experiments the ACSF contained 4 mM Sr2+ instead of CaCl2
and 4 mM MgCl2
so that the extracellular stimulation resulted in asynchronous presynaptic vesicle fusion. The stimulus strength was set so that the initial postsynaptic response was 50–100pA and the current amplitude and frequency of the asynchronous EPSCs occurring 400–900 ms post-stimulation was measured. Slices were treated with vehicle or 1-NA-PP1 from 2 DIV until the time of recording. Analysis of eEPSCs was performed using custom-written macros in IgorPro.
All animal experiments contained pups from multiple litters. All imaging analyses were done blind to condition. No data points were excluded in any experiment. Unpaired t-tests and 2-way ANOVA (for comparing effect of drug on AS EphB TKI neurons vs. wild-type) analyses were conducted using GraphPad Prism software. All tests are two-sided (standard). All error bars represent SEM.