Supplementary Information
The autism associated MET receptor tyrosine kinase engages early neuronal growth mechanism
and controls glutamatergic circuits development in the forebrain
Correspondent author:
Shenfeng Qiu, Ph.D.
[email protected]
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Supplementary Figure S1 (related to Figure 2). SU11274 and LY294002 inhibit Cdc42 activation.
a. Cdc42 activation, measured by GTP-bound Cdc42, was dose-dependently inhibited by MET kinase
activity inhibitor, SU11274 (10nM, 50nM). F(2,6) = 19.9. **p < 0.01. b. Cdc42-GTP was dose-dependently
reduced by the PI3K inhibitor LY294002 (5 M and 20 M). F(2,9) = 13.2, **p < 0.01.
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Supplementary Figure S2 (related to Figure 2). Dendritic and spine effects of RNAi-induced MET loss-offunction are rescued by the constitutively active Cdc42(V12).
a-d. Cultured cortical neurons were transfected with GFP alone, MET RNAi construct, the constitutively
active Cdc42(V12), MET RNAi + Cdc42(V12), and representative dendritic and spine morphology are
shown. e. MET RNAi reduced total dendritic branch length, which was rescued by co-transfection with
Cdc42(V12). F(3,24) = 4.75. *p < 0.05 compared with GFP; #p < 0.05; ns, not significant. f. MET RNAireduced dendritic spine density was reversed by co-transfection with Cdc42(V12). F(3,30) = 6.76. *p < 0.05,
**p < 0.01 compared with GFP; #p < 0.05; ns, not significant.
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Supplementary Figure S3 (related with Figure 5). Altered developmental MET signaling following single
neuron IUEP in the prefrontal cortex does not change neuronal membrane property.
One-way ANOVA reveals no significant change for resting membrane potentials (a), input resistance (b),
membrane capacitance (c) and action potential threshold (d) among GPF, MET OE and RNAi neurons.
Sample size was labeled on bar graph. (e) No change of L5 neuron spiking responses as a function of
current injections was observed among all the three groups (Two-way ANOVA, F(2, 156) = 0.04, p > 0.05 for
the treatment effect).
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Supplementary Materials and Methods
Mice
C57Bl6 mice were used for embryonic cortical/hippocampal neuron cultures, prefrontal cortex (PFC)
slice cultures and in utero electroporation studies. The day of vaginal plug detection was designated as
E0.5 and the day of birth as P0. Dorsal pallial-specific conditional Met mutant mice (Metfx/fx; Emx1cre,
designated as 'cKO') were generated and genotyped using previously described protocols 1. In this
model, cre-mediated excision of Met exon 16 leads to a loss-of-function 'kinase-dead' MET protein 2
(Figure 3a). Mice were kept in 12 h light/12 h dark cycle and had ad-lib access to food and water. All
procedures using mice were approved by the Institutional Animal Care and Use Committees of
University of Southern California and the University of Arizona and conformed to NIH guidelines. For all
studies, the experimenter was blind to genotypes.
DNA constructs, neuronal transfection and in utero electroporation (IUEP)
We used a Met cDNA vector that expresses the full ORF of mice Met gene (NM_008591, gift
from G. Vande Woude). The RNAi construct (19 nt reverse complimentary sequences) for Met was
cloned into pSuper vector 3 and its validity has been established previously 4. Cdc42 RNAi sequence
(GCAGTCACAGTTATGATTG) connected with a 9-nt spacer loop (TCTCTTGAA) and its reverse complement
sequence was cloned into pSuper (between bgl II and Hind III sites) and its efficiency was verified by cotransfection with Cdc42 cDNA in HEK293 cells (data not shown). Dominant negative Cdc42 (Cdc42-N17)
was a generous gift from D. Webb (Vanderbilt University). To visualize neuronal morphology, we cotransfected plasmids with pEGFP-C3 ('GFP') vector using calcium phosphate precipitation method 4. To
target the developing layer 5 (L5) PFC neurons in vivo and achieve altered Met gene expression, IUEP
experiments were carried out using time-pregnant mice at E13.5, similar to a previous report 5. Briefly,
the abdominal wall of the E13.5 TP mice (anesthetized with isoflurane) was opened by a ~2 cm incision.
The uterine horn and embryos were exposed and gently pulled outside the abdomen cavity. A sharp
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sterile needle filled cDNA mixture (with 0.05% fast green) was used to penetrate through the uterine
wall and the cortical plate of the developing brain to reach the lateral ventricle. ~1 l DNA was
pressure-injected into the lateral ventricles. Next, a tweezer-shaped electrode pair was positioned to
clamp the embryo heads in an orientation (~20° angle off para-sagittal plane and ~30° angle off the
horizontal plane, defined by an imaginary plane crossing the olfactory bulb and caudal cortical
hemisphere) so that the electrical field is targeting the developing right prefrontal area. Electrical
voltage pulses (40 V, 50 ms and 5 pulses, generated by an ECM 830 electroporator) were used to
electroporate DNAs into the developing PFC. The embryos were then returned to their original position
inside the dam. A sterile, size #6-0 fine monofilament medical suture was used to close the abdominal
wall. Bupivacaine local anesthetic (0.25 % gel) was applied topically at the incision site. Pups were
sacrificed at P23-25 and those with correct IUEP targeting were and used for electrophysiology
recording and LSPS mapping.
Primary cortical/hippocampal neuron culture and PFC slice cultures
Cortical neuron or hippocampal cultures were prepared from embryonic day E16.5 time pregnant
C57BL/6 mice. To facilitate immunocytochemistry (ICC) labeling, neurons were grown on 12-mm glass
coverslips at low densities (~10,000 cells/mm2 at plating), as described previously 4. High density
(~50,000 cells/mm2) hippocampal neurons grown in 6-well plates were used for protein collection.
Neurons were harvested at day in vitro (DIV) 5-28 for immunocytochemistry labeling or
electrophysiology recording. Only soma with a pyramidal shape and clear distinction of apical and basal
dendrites were selected for studies. For organotypic cultures of cortical slices containing PFC, postnatal
day 6-7 C57Bl6 mouse brains were dissected out in ice-cold ACSF, and 250 m-thick sagittal slices were
made with a vibratome (Leica V-1200S) under sterile conditions. Two slices adjacent to the mid-sagittal
line were obtained per mice, according to the anatomical landmarks of Allen Brain Atlas
(http://developingmouse.brain-map.org/static/brainexplorer). Slices were cultured on Millicell® cell
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culture inserts (Millipore, Billerica, MA) in DMEM/F12 medium supplemented with 10% FBS. HGF (50
ng/ml, Millipore) was added at 14 day in culture where necessary. Patch clamp recordings on L5 PFC
neurons were conducted at day 22-25.
Growth cone fraction isolation, biochemistry, Western blot and immunocytochemistry
We isolated growth cone-enriched fraction of postnatal day 0 mouse cortices using a method
reported by Pfenninger et al. 6 (Figure 1F). Briefly, 100 mg cortical tissue dissected from P0 C57Bl6 mice
was gently homogenized in a teflon-glass homogenizer in 600 μl 0.32 M sucrose containing 1 mM MgCl2
and 1 mM 2-[Tris(hydroxymethyl)methylamino]-1-ethanesulfonic acid-NaOH (pH adjusted to 7.3). The
homogenate was spun at 1,660 g for 15 min, and the low speed supernatant was loaded onto a
discontinuous sucrose density gradients (0.75, 1.0 M concentration), and further spun to equilibrium at
242,000 × g for 30 min using a Beckman SW 55 Ti swing bucket rotor. Thereafter, the ‘growth cone
particles’ fraction were collected at the low speed supernatant-0.75M sucrose interface, and subjected
to Western blot analysis. All the steps in isolating growth cone fractions were carried out on ice or at
4°C.
Western blot analysis of tissue/cells and immunocytochemistry (ICC) were performed and
quantified using standard methods, as reported previously 4. We micro-dissected PFC and hippocampus
regions and homogenized the tissue in cold NP40 cell lysis buffer (FNN0021, Life Technologies)
supplemented 1:50 protease inhibitor cocktail (Sigma P8340) and 1 mM PMSF. Total proteins were
quantified using a micro-BCA assay (Pierce) to ensure equal sample loading. Sample proteins are
separated on 9% SDS-polyacrylamide gels, transferred to PVDF membrane (Immobilon-P, Millipore),
incubated with primary antibodies diluted in 5% nonfat dry milk overnight at 4 °C, and followed by
incubation with HRP-conjugated secondary antibodies (Promega, Madison, WI) for 2 h at RT. Enhanced
chemiluminescence method (ECL Plus, GE Healthcare) was used to quantify the signal. The following
antibodies were used: from Santa Cruz Biotechnology, mouse anti-MET (sc-8057); from Millipore, goat
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anti-HGF (GF39), rabbit anti-GluA1 (AB1504) and anti-synapsin I (AB1543), mouse anti-GluN1 (MAB363
for Western blot; MAB1586 for ICC), rabbit anti-GluN2A (07-632), rabbit anti-GluN2B (05-920), mouse
anti-PSD95 (AB9708); from R&D systems, goat anti-HGF (AF276). From Cell Signaling Technology, rabbit
anti-GAPDH (5174), rabbit anti-pMET (Y1234/1235) (3077). To probe activated (GTP-bound) Cdc42, we
used a Cdc42 activation assay kit (Millipore, 17-441) according to manufacturer's directions. The kit
utilizes a glutathione-s-transferase fused with the p21-binding domain of p21-activated protein kinase
PAK1, which only binds and pulls down GTP-bound, activated form of Cdc42 or Rac1. The final dilution
of antibodies was between 1:1,000 and 1:2,000. Optical density of immunoreactive bands was
quantified by densitometry using Image J.
Acute brain slice preparation, drug treatment, patch clamp whole cell recording
To test the signaling competency of MET protein in developing mouse brain, we adopted an ex
vivo brain slice preparation. Acute coronal brain slices at postnatal day 9-10 containing PFC were cut
and kept alive in ACSF. Each slice was cut in the mid-sagittal line and separated into saline and HGF
treated groups. Slices were grouped into customized chamber designed to minimize turbulent ACSF flow
while aeronated with 95% O2 and 5% CO2 gas. Slices were treated with HGF (50 ng/ml, 30min) at 35°C in
the presence or absence of MET kinase inhibitor PHA665752 (200 nM), SU11274 (10 nM and 50nM), or
PI3K inhibitor Wortmanin (100 nM) or LY294002 (5M and 20M). PFC tissues from these slices were
then dissected, flash frozen, and subjected to Western blot analysis or small GTPase activation assay.
Miniature excitatory postsynaptic currents (mEPSCs) were recorded in cultured cortical neurons
and L5 PFC neurons in cultured slices using whole cell patch clamp technique. During recording, neurons
or slices were perfused with artificial cerebrospinal fluid (ACSF) containing (in mM), 126 NaCl, 2.5 KCl, 26
NaHCO3, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, and 10 glucose. ACSF was saturated with 95% O2 and 5% CO2
and perfused at 1.5–2 ml/min through the slice chamber. Neurons were visualized under an Olympus
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BX51WI microscope equipped with infrared DIC optics and an epifluorescent light source. The electrode
(4–6 MΩ) contains a potassium-based solution (in mM): 130 K-gluconate, 4 KCl, 2 NaCl, 10 HEPES, 4 ATPMg, 0.3 GTP-Na, 1 EGTA and 14 phosphocreatine(pH 7.2, 295 mOsm). In experiments where
AMPA/NMDAR current ratio and ifenprodil sensitivity were measured, the patch electrodes contained
(in mM): 125 Cs gluconate, 5 tetraethylammonium–Cl, 10 Hepes, 8 NaCl, 5 QX314-Cl, 4 Mg2+-ATP, 2.5
CsCl, 0.3 Na3GTP, 0.2 EGTA, 10 phosphocreatine, and adjusted to pH 7.2, 280–290 mOsm. For all whole
cell experiments, series resistance (Rs) was monitored throughout recordings; only stable (<15% change)
cells with Rs < 25 MΩ throughout the recordings were included. All chemical ligands used in this study,
including Wortmanin, PHA665732, SU11274, LY294002, ifenprodil, R-CPP and MNI-caged glutamate
were purchased from Tocris/R&D Systems.
Laser scanning photostimulation (LSPS) for cortical circuit mapping
For LSPS mapping of prefrontal circuits, essentially the same protocol was reported previously 7,
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. Pups born to IUEP dams were sacrificed at P22-25. Sagittal slices containing the right PFC were made
(usually 1-2 slices obtained per mouse). Individual GFP-expressing PFC L5 neurons were selected for
patch clamp recording and LSPS mapping based on anatomical landmarks delineated by the Allen Brain
Atlas (prelimbic area, layer 5). Neurons were selected under epifluorescence illumination and targeted
for recording under DIC.
Electrophysiological signals were amplified with a Multiclamp 700B amplifier (Molecular Devices)
and acquired with data acquisition boards (BNC 6259, National Instruments) under control of Ephus
software (http://www.ephus.org). Signals were digitized and acquired at 10 kHz. Passive membrane
properties were calculated based on current responses to negative voltage steps. Intrinsic properties
were measured in current-clamp mode by injecting current steps (1-s duration, −100 to +500 pA in 50pA
increments). Traces were analyzed off-line to calculate the current -firing frequency relationships. LSPS
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mapping on slices was performed at RT in a customized slicechamber mounted on a motorized stage
(Sutter MPC-78). The chamber was perfused by modified ACSF with elevated concentrations of
magnesium and calcium containing (in mM): 126 NaCl, 2.5 KCl, 26 NaHCO3, 4 CaCl2, 4 MgCl2, 1.25
NaH2PO4, and 10 glucose at 1.5–2 ml/min. 5 μM R-CPP and 0.2 mM MNI-caged glutamate were added to
circulating ACSF. Neuronal somata selected for recording were at least 50 μm below the surface to
minimize truncation of dendritic structures. LSPS mapping was performed through a 4× objective lens
(NA 0.16; Olympus) and 20 mW, 1-ms UV laser (355 nm; DPSS Lasers) pulses were scanned onto the slice
after passing an electro-optical modulator (Conoptics) and a mechanical shutter (Uniblitz). Neuronal
responses to laser uncaging/mapping were analyzed offline with custom Matlab routines.
Neuronal morphological analysis
To reveal neuronal dendritic morphology in Met cKO and littermate control slices, we used a rapid Golgi
staining kit (FD Neurotechnologies). The P35-38 brains were fixed, sectioned into 250 m slices, and
mounted on slides according to the manufacture's protocol. Dendritic arbors were manually traced
using Neurolucida (Microbrightfield). Morphometric parameters were extracted using Neurolucida
explorer, similar to that described previously 9. To reconstruct neuronal spine morphology in L5 PFC
neurons from Met cKO mice and their littermate controls, we first injected 50 nanoliter retrograde red
fluorescent latex microspheres ('beads', Lumafluor Inc.) into left dorsal striatum, then prepared PFC slice
at least 24 h later. L5 beads-positive CS neurons were dialyzed with 1% biocytin dissolved in the
potassium-based internal electrode solution (500 pA holding current for 10 min). Slices were then fixed
by 4% PFA overnight followed by avidin-Alexa488 (Life technologies) conjugation for 24 h. High
resolution Z stacks were acquired from secondary apical branches 150-300 m way from soma using a
Zeiss 710 confocal microscope (63x oil objective, NA 1.4). Dendritic segment images were acquired with
2x digital zoom, a frame size of 1024×1024 pixels, and 0.2 μm Z interval. This yields a voxel size of
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0.069×0.069×0.2 cubic micron. The confocal files were imported into Imaris (Bitplane, V7.01) software
and visualized in the ‘surpass’ view. The Filament module with Autopath (no loops) was used to for 3D
rendering of each dendritic segment. Renderings were subsequently edited manually to include or
exclude spines that are misidentified. We used Imaris to measure spine parameters and classify them
into 'filopodia', 'stubby', 'mushroom' and 'long thin' categories using Imaris default spine classification
module 10.
Statistical Analysis
Quantitative results are expressed as mean ± SEM. Data was processed using Microsoft Excel, Matlab
and GraphPad Prism 5.0. Sample size was estimated by power analysis using an R script that takes prespecified effect size, type I and II errors as input arguments. The same algorithm was used to estimate
sample size of mice. Student t test or one-way/two-way analysis of variations (ANOVA) was used when
data passed normality test. Potential data outliers are identified as those differ at least three standard
deviations from the mean and excluded from analysis where applicable. For IUEP experiments, mice
were grouped randomly. Sample size n represents number of independent treatments obtained from at
least three mice. Kolmogorov-Smirnov (K-S) test was used to compare cumulative distribution of mEPSC
amplitude and inter-event intervals. Spine head size (non-normal distribution) comparison was made
using non-parametric Mann-Whitney test. Dunnett’s multiple comparisons or Bonferroni post hoc test
was used for between-group comparisons after ANOVA reveals overall significance. p < 0.05 was
considered statistically significant.
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