www.sciencemag.org/cgi/content/full/science.1252514/DC1

Supplementary Materials for

Retrograde Semaphorin Signaling Regulates Synapse Elimination in the

Developing Mouse Brain

Naofumi Uesaka, Motokazu Uchigashima, Takayasu Mikuni, Takanobu Nakazawa,

Harumi Nakao, Hirokazu Hirai, Atsu Aiba, Masahiko Watanabe, Masanobu Kano*

*Corresponding author. E-mail: mkano-tky@m.u-tokyo.ac.jp

Published 15 May 2014 on Science Express

DOI: 10.1126/science.1252514

This PDF file includes:

Materials and Methods

Figs. S1 to S22

Tables S1 to S3

Full Reference List

Materials and methods

Animals

Sprague-Dawley (SD) rats and C57BL/6 mice were used (SLC JAPAN). All experiments

were performed in accordance with the guidelines set down by the experimental animal

ethics committees of the University of Tokyo, Hokkaido University, and the Japan

Neuroscience Society. The coding region of enhanced green fluorescent protein (GFP) was

inserted into the pL7ΔAUG vector (20, 21) and then the L7-GFP transgene was injected

into fertilized eggs to generate L7-GFP transgenic mice.

Isolation of Purkinje cells using a fluorescence-activated cell sorter (FACS)

The method has been described previously (21), but was modified slightly. Briefly, the

cerebellum was removed from L7-GFP transgenic mice at postnatal day (P)4, P6, P7, P9,

P13, P15, and P18. Cubes of cerebella were digested for 10 min at 37°C with 90 U of

papain (Worthington, NJ, USE) in a dissociation solution consisting of Ca2+

-free Hank’s

balanced salt solution (HBSS) containing 0.2 mg/ml DL-cysteine HCl, 0.2 mg/ml bovine

serum albumin, 1 mM HEPES, 1.5 mM MgSO4 and 5 mg/mL glucose (pH 7.4). The

enzymatic reaction was stopped by the addition of dissociation solution containing 5 U/mL

DNAase I. Tissues were triturated mildly through wide-bore and fine-tipped pipettes. After

centrifuging, the supernatant was removed and the cells were resuspended in Ca2+

- and

Mg2+

-free PBS. The cells were then filtered through a 40-μm nylon mesh. Cell sorting was

performed with a FACS EPICS ALTRA (Beckman Coulter, Inc., CA, USA). For sorting

Purkinje cells, cells were passed through a 70-μm nozzle at a rate of 10 000–20 000 events/s

at first sorting. After the first sorting, the passed cells were centrifuged and resuspended.

The resuspended cells were passed at a rate of 1000 events. The sort decision was based on

the measurements of forward scatter (FSC) and GFP fluorescence. Thresholds were

optimized by comparing the percentage of Purkinje cells before and after sorting.

Profiling of genes expressed in Purkinje cells

Total RNA was extracted from the isolated cells and used as a template in the GeneChip

Mouse Genome 430 2.0 DNA microarray (Affymetrix, CA, USA). Raw image files were

processed using Affymetrix GCOS and Microarray Suite (MAS) 5.0. The MAS 5.0 software

includes a detection algorithm that uses probe pair (perfect match/mismatch) intensities to

generate a detection P-value, which is then used to determine whether a transcript is present

or absent. Intensity data from each chip were normalized to a target intensity value of 500,

and expression data and absent/present calls for individual probe sets were determined. For

the analysis in the present experiment, the default threshold for a present call was used.

Candidate genes for screening were chosen by picking genes reported as membrane or

secreted molecules, or by sorting genes using the keywords “membrane” or “extracellular

space.”

Organotypic coculture of the cerebellum and medulla oblongata

The olivo-cerebellar cocultures were prepared as described previously (7, 8). In brief, the

ventral medial portion of the medulla (containing inferior olivary neurons) was dissected

from rats on embryonic day (E)15. Cerebellar slices (250 µm thick) were dissected from the

vermis of P10 mice. A block of the medulla was plated at the ventricular side of the

cerebellar slice on a membrane filter (Millicell-CM PICMORG50, Millipore, MA, USA),

which was coated with rat-tail collagen. The culture medium consisted of 50% MEM, 25%

horse serum (STEMCELL Technologies, BC, Canada), 2.5% Hank’s balanced salts solution

(Nacalai Tesque, Kyoto, Japan), 3 mM GlutaMAX (Gibco, NY, USA), 10 µM Mifepristone

(Ru486, TOCRIS, Bristol, UK), 5 mg/ml glucose, and 2% B27 supplements (Gibco). The

cultures were maintained at 37°C in an environment of humidified 95% air and 5% CO2.

Half of the culture medium was exchanged with fresh medium every other day.

Preparation of viral vector constructs

Virus vectors were constructed as previously described (7). Briefly, the vectors were

designed to express GFP, microRNA (miRNA), and/or cDNA under the control of the

murine embryonic stem cell virus (MSCV) (pCL20c-MSCV) or under the control of a

truncated L7 promoter (pCL20c-trL7) (23). The cDNA for Sema3A, Sema7A, PlxnA4, or

ItgB1 was obtained by RT-PCR of a cDNA library from P10 mouse cerebellum or medulla.

The cDNA for PlxnC1 was purchased from Origene (MD, USA). Each cDNA was

subcloned into pCL20c-trL7 or pCL20c-MSCV. The BLOCK-iT Pol II miR RNAi

expression vector kit (Invitrogen, CA, USA) was used for vector-based RNA interference

(RNAi) analysis. The following engineered microRNAs were designed according to the

BLOCK-iT Pol II miR RNAi Expression Vector kit guidelines (Invitrogen):

5'-TGCTGTATAGGTGCAGATTGGATGGAGTTTTGGCCACTGACTGACTCCATCCA

CTGCACCTATA-3' and

5'-CCTGTATAGGTGCAGTGGATGGAGTCAGTCAGTGGCCAAAACTCCATCCAATC

TGCACCTATAC-3' for Sem3A-microRNA;

5'-TGCTGAATACACGCAGACAGCTGAGTGTTTTGGCCACTGACTGACACTCAGC

TCTGCGTGTATT-3' and

5'-CCTGAATACACGCAGAGCTGAGTGTCAGTCAGTGGCCAAAACACTCAGCTGT

CTGCGTGTATTC-3' for Sema7A-microRNA;

5'-TGCTGAATCCAAGTTTCCAGATATGCGTTTTGGCCACTGACTGACGCATATCTA

AACTTGGATT-3' and

5'-CCTGAATCCAAGTTTAGATATGCGTCAGTCAGTGGCCAAAACGCATATCTGGA

AACTTGGATTC-3' for ItgB1-microRNA;

5'-TGCTGATAAACAGACGTCAGGTCAGAGTTTTGGCCACTGACTGACTCTGACCT

CGTCTGTTTAT-3' and

5'-CCTGATAAACAGACGAGGTCAGAGTCAGTCAGTGGCCAAAACTCTGACCTGA

CGTCTGTTTATC-3' for PlxnC1-microRNA;

5'-TGCTGAAAGGAAGGCTTCTGAGACAGGTTTTGGCCACTGACTGACCTGTCTC

AAGCCTTCCTTT-3' and

5'-CCTGAAAGGAAGGCTTGAGACAGGTCAGTCAGTGGCCAAAACCTGTCTCAG

AAGCCTTCCTTTC-3' for PlxnA4-microRNA;

5'-TGCTGTCAACTTGTAGACACGATTGAGTTTTGGCCACTGACTGACTCAATCGT

CTACAAGTTGA-3' and

5'-CCTGTCAACTTGTAGACGATTGAGTCAGTCAGTGGCCAAAACTCAATCGTGTC

TACAAGTTGAC-3' for PlxnA2-microRNA;

5'-TGCTGTTACATTGTAGCCCAGAGACCGTTTTGGCCACTGACTGACGGTCTCTG

CTACAATGTAA-3' and

5'-CCTGTTACATTGTAGCAGAGACCGTCAGTCAGTGGCCAAAACGGTCTCTGGG

CTACAATGTAAC-3' for FAK-microRNA. These oligonucleotides were subcloned into a

pCL20c-trL7 vector or into a pCL20c-MSCV vector. The QuikChange Lightning

site-directed mutagenesis kit (Agilent Technologies, CA, USA) was used to generate

RNAi-resistant forms of Sema3A, Sema7A, PlxnA4, PlxnC1, and ItgB1 (Sema3A-rescue,

Sema7A-rescue, PlxnA4-rescue, PlxnC1-rescue and ItgB1-rescue), which harbor sense

mutations (no alteration of amino acid codons) and constitutively active cofilin

(cofilin-CA). Sema3A-rescue, Sema7A-rescue, PlxnA4-rescue, PlxnC1-rescue,

ItgB1-rescue, FAK-rescue, or cofilin-CA were either linked in-frame to mOrange2

interposed by a picornavirus “self-cleaving” P2A peptide sequence to enable efficient

bicistronic expression, or fused to a FLAG tag, and then subcloned into pCL20c-trL7 or

pCL20c-MSCV. Scrambled control miRNA for PlxnC1, ItgB1, or PlxnA4 was designed by

shuffling the recognition region sequences. A BLAST search confirmed that the scrambled

sequences had no target gene. All constructs were verified by DNA sequencing.

Virus preparation and infection

The detailed procedure for viral vector production has been described previously (7, 8). For

in vitro virus infection, cultured Purkinje cells were infected at 0 or 1 day in vitro (DIV) by

adding 0.5 µl (0.2–1.0 × 105 TU) of viral solution per coculture directly on the

slices. For in

vivo virus infection into the cerebellum, 4–5 μl (4–5 × 105 TU) of viral solution were

injected into the cerebellar vermis of C57BL/6 mice at P1–P2. For in vivo virus infection

into the inferior olivary neurons, 1–2 µl (1–2 × 105 TU) of viral solution were injected into

the ventral medial portion of the medulla at P0–P2.

Electrophysiological recordings from Purkinje cell in cocultures

To obtain electrophysiological recordings from Purkinje cells in coculture, most of the

medullary explant was cut from the coculture to reduce responses elicited by spontaneous

firing of inferior olivary neurons. The culture preparations were recovered for at least 30

min at room temperature by incubation in a reservoir chamber bathed in a solution of 125

mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 1.25 mM NaH2PO4, 26 mM

NaHCO3, and 20 mM glucose bubbled with 95% O2 and 5% CO2. The preparations were

transferred to a recording chamber located on the stage of an Olympus BX51WI

microscope. The recording chamber was continuously perfused with oxygenated bath

solution supplemented with picrotoxin (TOCRIS; 0.1 mM) to block inhibitory synaptic

transmission. Whole-cell recordings were made from visually identified or fluorescent

protein-positive Purkinje cells using upright and fluorescence microscopes at 32°C. The

resistance of the patch pipettes was 1.5–2.5 MΩ when filled with an intracellular solution

comprising 60 mM CsCl, 10 mM Cs D-gluconate, 20 mM TEA-Cl, 20 mM BAPTA, 4 mM

MgCl2, 4 mM ATP, and 30 mM HEPES (pH 7.3, adjusted with CsOH). The pipette access

resistance was compensated by 70%. The holding potential for recording climbing

fiber-mediated excitatory postsynaptic currents (CF-EPSCs) was -30 mV (corrected for the

liquid junction potential). The climbing fiber innervation pattern was examined after 15–18

days by taking whole-cell recordings in knockdown and uninfected control Purkinje cells in

the same coculture. To activate climbing fibers, four tungsten electrodes (3.5–4.5 MΩ,

Catalog #UEWMGCSEKNNM, FHC, Maine, USA) were placed in the remaining portions

of the medullary explants. Single or multiple steps of CF-EPSCs were elicited in a given

Purkinje cell when the intensity of stimulation in the medullary explant was increased

gradually. The number of climbing fibers innervating the recorded Purkinje cell was

estimated based on the number of discrete CF-EPSC steps elicited in that Purkinje cell (12).

Electrophysiological recordings from Purkinje cells in acute cerebellar slices

Parasagittal cerebellar slices (250 μm) were prepared from mice (12) from P6 to P60. The

bathing and intracellular solutions were the same as those used for recording coculture

preparations. The recording chamber was continuously perfused with oxygenated bath

solution supplemented with picrotoxin (0.1 mM). The holding potential for recording

CF-EPSCs was -20 mV and that for recording EPSCs induced by parallel fiber stimulation

was -80 mV (corrected for the liquid junction potential). To record CF-EPSCs, stimuli

(duration, 0.1 ms; amplitude, 0–30 µA) were applied at 0.2 Hz. Climbing fibers were

stimulated in the granule cell layer 20–100 μm from the Purkinje cell soma. The number of

climbing fibers innervating the recorded Purkinje cell was estimated according to the

number of discrete CF-EPSC steps (12). To examine the effects of knocking down PlxnA4,

PlxnC1, or ItgB1 in climbing fibers, Purkinje cells were sampled at locations in which

virus-infected climbing fibers were rich (GFP-positive regions) and from locations in which

transfected climbing fibers were absent (GFP-negative regions). Climbing fiber innervation

patterns were compared between GFP-positive and GFP-negative regions in the same

slices.

Immunohistochemistry

Mice were placed under deep pentobarbital anesthesia (100 μg/g of body weight,

intraperitoneal injection), perfused with 4% paraformaldehyde in 0.1 M phosphate buffer,

and then processed to obtain parasagittal microsliced sections (100 μm in thickness). After

permeabilization and blockade of nonspecific binding, the following antibodies were

applied overnight at 4°C: a mouse monoclonal antibody against calbindin D-28K (diluted

1:3,000, Sigma, St. Louis, MO) to visualize Purkinje cells; an antibody against guinea pig

vesicular glutamate transporter VGluT2 (1:200, Frontier Institute, Hokkaido, Japan), to

label climbing fiber terminals; an antibody against rabbit glutamate receptor δ2 (GluD2)

(1:200, Frontier Institute, Hokkaido, Japan); an antibody against rabbit metabotropic

glutamate receptor 1 (mGluR1) (1:200, Frontier Institute, Hokkaido, Japan); an antibody

against goat Sema3A (1:100, Santa Cruz, CA, USA); or an antibody against GFP (1:1000,

Nacalai Tesque, Kyoto, Japan). After incubation with secondary antibodies (an anti-rat

Alexa Fluor 488 antibody or an anti-mouse Cy5 antibody, Jackson; 1:300; or an anti-rabbit

Cy3 antibody, Jackson; 1:300), the immunolabeled sections were washed and then

examined under a confocal laser scanning microscope (FV10i and FV1200, Olympus).

Evaluation of knockdown efficacy

HEK 293T cells in a 24-well dish were transfected with an RNAi knockdown vector

(Sema3A-knockdown, Sema7A-knockdown, PlxnA4-knockdown or ItgB1-knockdown)

and mOrange2-fused cDNA (Sema3A-mOrange, Sema7A-mOrange, PlxnA4-mOrange or

ItgB1-FLAG) using X-tremeGENE 9 reagents. One day later, the cells were observed under

a confocal laser scanning microscope (LSM 510, Zeiss). For PlxnC1, the RNAi knockdown

vector (PlxnC1-knockdown) and PlxnC1 cDNA were transfected into HEK 293T cells. One

day later, the cells were fixed. After permeabilization, blocking, and application of primary

(rabbit anti-PlxnC1, rabbit anti-DsRed, or mouse anti-FLAG) and secondary (donkey

anti-rabbit Cy3; Jackson; 1:300, or donkey anti-mouse Cy3, Jackson; 1:300) antibodies, the

fluorescence signals were examined under a confocal laser scanning microscope (LSM 510,

Zeiss).

In situ hybridization

Mouse cDNA fragments of Sema3A (nucleotides 502–2369; GenBank accession number

NM_001243072), Sema7A (8–2017, NM_011352), PlxnA4 (1376–2965, NM_175750.3),

PlxnA2 (556–2496, NM_008882.2), PlxnC1 (316–1304, NM_018797), ItgB1 (528–2188,

NM_010578), VGluT2 (934–2060; BC038375), and 67 kDa-glutamic acid decarboxylase

(GAD67; 1036–2015; NM_008077) were used. Digoxigenin (DIG)- or fluorescein-labeled

cRNA probes were prepared for simultaneous detection of multiple mRNAs by fluorescence

in situ hybridization (24). In brief, fresh frozen sections were hybridized with a mixture of

DIG- or fluorescein-labeled cRNA probes. After a stringent posthybridization wash, DIG and

fluorescein were detected using a two-step method as follows: the first detection was

performed using a peroxidase-conjugated anti-fluorescein antibody (Invitrogen) plus the

FITC-TSA plus amplification kit (PerkinElmer, MA, USA); the second detection was

performed with a peroxidase-conjugated anti-DIG antibody (Roche Diagnostics, Mannheim,

Germany) and the Cy3-TSA plus amplification kit (PerkinElmer). TOTO3 (Invitrogen) was

used for fluorescent nuclear counterstaining.

In utero electroporation

For experiments involving double knockdown of Sema3A or Sena7A in Purkinje cells and

of PlxnA4, PlxnC1 or ItgB1 in climbing fibers (Fig. 4 G to L), miRNA for Sema3A- or

Sema7A-knockdown was introduced into Purkinje cells using in utero electroporation (25,

26). Briefly, pregnant mice at embryonic day (E)11.5 or E12.5 were deeply anesthetized with

an intraperitoneal injection of sodium pentobarbital (Somnopentil; Kyoritsu Seiyaku Co.,

Tokyo, Japan). Electric pulses (40–50 V for 50 ms, five times at 950 ms intervals) were

delivered via forceps-shaped electrodes (CUY650P2 or CUY650P3, Unique Medical Imada,

Aichi, Japan) connected to an electroporator (CUY21, Nepa Gene, Chiba, Japan). Plasmids

were dissolved in water. The plasmid vector, pCL20c-trL7, was used to knockdown the

expression of Sema3A and Sema7A in Purkinje cells.

Protein preparation and expression analysis

Cerebella from wild-type and mGluR1 knockout mice (P14) were lysed in TNE buffer (1%

(w/v) Nonidet P-40, 50 mM Tris-Cl (pH 7.5), 100 mM NaCl, 5 mM EDTA, 0.2 mM

Na3VO4), the lysates resolved by sodium dodecyl sulfate polyacrylamide gel

electrophoresis, and the proteins transferred to polyvinylidene difluoride membranes

(Bio-Rad, CA, USA). The membranes were blocked with 3% bovine serum albumin

(Fraction V) and 0.1% Tween-20 in Tris-buffered saline for 10 min and blotted with the

appropriate primary antibodies, followed by the appropriate secondary antibodies. The

following commercially available antibodies were used: anti-mGluR1 (Frontier Institute,

Hokkaido, Japan), anti-semaphorin 7A (R&D Systems Inc., MN, USA), anti-tubulin (Cell

Signaling, MA, USA), anti-rabbit-IgG-AP (Santa Cruz, TX, USA), anti-mouse-IgG-AP

(Santa Cruz), anti-rabbit-IgG-HRP (GE Healthcare, NJ, USA), and anti-mouse-IgG-HRP

(GE Healthcare). Horseradish peroxidase-Renaissance Plus Reagent (Perkin-Elmer, MA,

USA) or CDP-star (Roche, Mannheim, Germany) were used to visualize the

immunoreactive proteins. Data acquisition and analysis were performed using LAS 4000

(GE Healthcare).

Surface biotinylation assay

The receptor biotinylation assay was performed using cleavable biotin, as described

previously (22). Briefly, the surface proteins expressed by cerebellar slices were

biotinylated with 1 mg/ml sulfo-NHS-SS-biotin (Pierce, IL, USA) for 20 min at 4°C. To

collect the surface proteins, the cells were lysed with lysis buffer (20 mM Tris-Cl [pH 7.5],

100 mM NaCl, 5 mM EDTA, and 1% NP-40) and the biotinylated proteins were

precipitated with NeutrAvidin resin (Pierce). Samples were subjected to immunoblotting as

described above.

Statistical analysis

Data were expressed as the mean ± SEM. The Mann–Whitney U test was used when two

independent data sets were compared (except for the immunoblotting data). Student’s t-test

was used to compare the immunoblotting data. Differences between groups were judged to

be significant when P < 0.05. For multiple comparisons, the Steel-Dwass test and two-way

ANOVA were used as indicated in the text. All statistical analyses were performed using

JMP Pro software (NC, USA).

Fig. S1. Strategy for RNA interference (RNAi) screening of retrograde signaling

molecules required for climbing fiber synapse elimination in olivo-cerebellar

cocultures.

Fig. S2. Purification of Purkinje cells from the cerebella of L7-GFP transgenic mice.

(A) Bright field and epifluorescent images of whole brain from an L7-green fluorescent

protein (GFP) transgenic mouse at P9. (B, C) Confocal micrographs of cerebellar sections

of postnatal day (P)4 (B) and P16 (C) L7-GFP transgenic mice. (D, E) Purification of

GFP-expressing Purkinje cells by cell sorting. (D) Fluorescence histograms for cerebellar

cells at first (left) and second (right) sorting. (E) A representative photomicrograph showing

Purkinje cells sorted from cerebellar cells from P9 L7-GFP transgenic mice. Note that most

cells express GFP. Scale bar, 50 µm.

Fig. S3. Efficacy of Sema3A- or Sema7A-knockdown by respective microRNA vectors.

(A) Double immunostaining for mOrange (red) and green fluorescence protein (GFP, green)

in HEK 293T cells transfected simultaneously with Sema3A-mOrange and a

GFP-expression vectors (left, “Control”), with Sema3A-mOrange and GFP-expression

vectors together with a microRNA vector against Sema3A (middle, “Sema3A-KD”), or

with an RNA interference (RNAi)-resistant Sema3A-mOrange vector and a

GFP-expression vector together with a microRNA vector against Sema3A (right,

“Sema3A-RES”). Scale bar, 30 µm. (B) Fluorescence intensity of mOrange relative to that

of GFP in control (white column, n = 8), Sema3A-KD (green column, n = 6), and

Sema3A-RES (light green column, n = 11) cells. (C) Similar to (A), but for

Sema7A-knockdown (Sema7A-KD) and Sema7A-rescue (Sema7A-RES). (D) Fluorescence

intensity of mOrange relative to that of GFP in control (n = 9), Sema7A-KD (orange

column, n = 7), and Sema7A-RES (light orange column, n = 6) cells. * P < 0.05 and ** P <

0.005 (Steel-Dwass test).

Fig. S4. Expression of mRNAs for Sema3A and Sema7A in the developing mouse

brain.

(A) Coronal sections of mouse brain showing expression of Sema3A and Sema7A mRNA

at postnatal day (P)7 and P14. SP: Fluorescent signals using a sense probe. (B–E) Triple

fluorescence in situ hybridization detecting mRNAs for Toto3 (blue, nuclei), GAD67 (green,

inhibitory neurons, including Purkinje cells) and either Sema3A (B, C) or Sema7A (D, E) in

the cerebellum at P7 (B, D) and P14 (C, E). Sema3A and Sema7A mRNAs are present in

the somata of Purkinje cells (GAD67 mRNA-positive large cells in the Purkinje cell layer)

at both P7 and P14. Arrows show the same positions in the left and right panels. EGL:

external granular layer, IGL: internal granular layer, pia: pial surface. Scale bars, 1 mm for

(A), 20 µm for (B–E).

Fig. S5. Acceleration of climbing fiber synapse elimination during postnatal

development without persistent effects in adulthood in Sema3A-knockdown Purkinje

cells.

(A–E) Frequency distribution of the number of climbing fibers innervating each Purkinje

cell in control (white columns) and Sema3A-KD (green columns) cells from postnatal day

(P)6–P7 (A, n = 21 for the controls; n = 21 for Sema3A-KD), P8–P11 (B, n = 38 for the

control; n = 37 for Sema3A-KD), P12–P14 (C, n = 55 for the controls and n = 63 for

Sema3A-KD), P15–P18 (D, n = 46 for the controls and n = 53 for Sema3A-KD), and

P21–P30 (E, n = 53 for the controls and n = 58 for Sema3A-KD). *P < 0.05 and **P <

0.005. ns indicates no significant difference (Mann–Whitney U test).

Fig. S6. Morphological evidence for the acceleration and impairment of climbing fiber

synapse elimination from somata of Purkinje cells by Sema3A-knockdown and

Sema7A-knockdown, respectively.

(A–D) Digital representation of confocal microscopic images showing immunoreactivity of

calbindin (blue, Purkinje cells), vesicular glutamate transporter VGluT2 (red, climbing

fiber terminals), and enhanced green fluorescent protein, EGFP (green) in control (A) and

Sema3A-KD (B) Purkinje cells in the same tissue slice from postnatal day (P)14 mouse

cerebellum, and in control (C) and Sema7A-KD (D) Purkinje cells in the same slice from

the cerebellum of a P21 mouse. White arrows indicate VGluT2 terminals that contact

Purkinje cells.

Fig. S7. Impairment of climbing fiber synapse elimination after P15 (with persistent

defect into adulthood) in Purkinje cells of a Sema7A-knockdown.

(A–E) Frequency distribution of the number of climbing fibers innervating each Purkinje

cell in the control (white columns) and Sema7A-KD (orange columns) during postnatal day

(P)8–P11 (A, n = 23 for the control; n = 21 for Sema7A-KD), P12–P14 (B, n = 18 for the

control; n = 22 for Sema7A-KD), P15–P18 (C, n = 38 for the control; n = 45 for

Sema7A-KD), P21–P30 (D, n = 57 for the control; n = 63 for Sema7A-KD) and P40–P60

(E, n = 32 for the control; n = 34 for Sema7A-KD). *P < 0.05, **P < 0.01, and ***P <

0.005. ns indicates no significant difference (Mann–Whitney U test).

Fig. S8. Efficacy of knocking down mGluR1 or GluD2 using microRNA vectors.

(A, B) Triple immunostaining for calbindin (blue), green fluorescent protein (GFP, green),

and metabotropic glutamate receptor 1 (mGluR1, red) in control (A) and

mGluR1-knockdown (B) Purkinje cells in the same tissue slice. (C, D) Similar to (A) and

(B), but showing staining for glutamate receptor δ2 (GluD2, red) in control (C) and

GluD2-knockdown (D) Purkinje cells in the same slice. Scale bars, 20 µm for (A–D).

Fig. S9. Effects of Sema3A- or Sema7A-knockdown on Purkinje cell synaptic

responses from parallel fibers and inhibitory interneurons.

(A, B) Representative traces (top) and the averaged input-output relationships (bottom)

between excitatory postsynaptic currents (EPSCs) induced by stimulation of parallel fibers

in control (white circles, n = 24), Sema3A-KD (green circles, n = 23), and Sema3A-RES

(light green circles, n = 12) Purkinje cells (A), and in control (n = 32) and Sema7A-KD

(orange circles, n = 20) Purkinje cells (B). The holding potential was -80 mV. Scale bars,

0.1 nA, 5 ms for (A) and 0.2 nA, 5 ms for (B). Asterisks indicate significant differences

when compared with the control (*P < 0.05, two-way ANOVA; Steel-Dwass test). (C–H)

Sample traces of miniature inhibitory postsynaptic currents (mIPSCs) (C, F) and summary

bar graphs showing the mean frequency (D, G) and amplitude (E, H) of mIPSCs for control

(white columns, n = 9) and Sema3A-KD (green columns, n = 9) cells (D, E) and for control

(n = 9) and Sema7A-KD (orange columns, n = 9) cells (E, H). Scale bars, 20 pA and 10 ms.

Fig. S10. Expression of mRNAs for PlxnA2, PlxnA4, PlxnC1, and ItgB1 in inferior

olivary neurons during postnatal development.

(A, B) Triple fluorescence in situ hybridization analysis of mRNAs for Toto3 (blue, nuclei),

the vesicular glutamate transporter VGluT2 (green, inferior olivary neurons), and PlxnA2

(top in A, red), PlxnA4 (bottom in A, red), PlxnC1 (top in B, red), or ItgB1 (bottom in B,

red) in the inferior olive at postnatal day (P)7 (top) and P14 (bottom). Signals for PlxnA2,

PlxnA4, PlxnC1, and ItgB1 mRNA are clearly present in VGluT2 mRNA-positive cells at

both P7 and P14. Arrows indicate the same positions in the left and right panels. Scale bars,

20 µm.

Fig. S11. Efficacy of PlxnA4, PlxnC1, ItgB1 or PlxnA2 knockdown using microRNA

vectors.

(A, C, E, G) HEK 293T cells were transfected with the indicated constructs and then

double immunostained with anti-GFP and anti-PlxnC1 antibodies to detect PlxnC1 (C) or

with an anti-FLAG antibody to detect PlxnA4, ItgB1, and PlxnA2 (A, E, G). Scale bars, 30

µm. (B, D, F, H) Fluorescence intensity of PlxnA4, PlxnC1, ItgB1 or PlxnA2 relative to

that of GFP in control cells (white columns, n = 13 for B; n = 17 for D; n = 17 for F; and n

= 12 for H), knockdown (KD) cells (green column, n = 11 for B; orange column, n = 13 for

D; yellow column, n = 16 for F; and blue column, n = 12 for H), cells expressing scramble

miRNAs (SCR) (light green column, n =11 for B; light orange column, n = 14 for D; and

light yellow column, n =15 for F), and cells expressing a microRNA (miRNA)-resistant

form of cDNA (RES) (deep green column, n = 12 for B; brown column, n = 14 for D; dark

yellow column, n = 9 for F). *P < 0.05, ** P < 0.01, and *** P < 0.005 (Steel-Dwass test).

Fig. S12. Gene delivery into inferior olivary neurons in vivo.

(A) Expression of enhanced green fluorescent protein (EGFP) in inferior olivary neurons at

postnatal day (P)10. Viruses expressing EGFP and one of the microRNAs (miRNAs)

against Sema3A and Sema7A receptors were injected into the ventral medial medulla of

P0–P1 mice. (B) EGFP-labeled climbing fibers in the cerebellum. Scale bars, 10 µm.

Fig. S13. Rescuing the effect of PlxnA4-knockdown, and enhanced parallel fiber to

Purkinje cell synaptic transmission by PlxnA4 knockdown.

(A) Examples of climbing fiber-mediated excitatory postsynaptic currents (CF-EPSCs) in

Purkinje cells associated with climbing fibers expressing microRNA (miRNA) for PlxnA4

together with a miRNA-resistant form of PlxnA4 (plxnA4-RES). Scale bars, 0.5 nA and 5

ms. (B) Frequency distribution of the number of climbing fibers innervating each control

(white columns, n = 26) and PlxnA4-RES (green columns, n = 25) Purkinje cells at

postnatal day (P)11–P13. ns indicates no significant difference (Mann–Whitney U test). (C)

Representative traces (top) and averaged input-output relationships (bottom) between

EPSCs induced by parallel fiber stimulation in the control (white circles, n = 28),

PlxnA4-knockdown (PlxnA4-KD, green circles, n = 11) and PlxnA4-scramble

(PlxnA4-SCR, light green circles, n = 14) Purkinje cells. The holding potential was -80 mV.

Scale bars, 0.1 nA and 5 ms.* P < 0.05, ** P < 0.01, and *** P < 0.005 (two-way ANOVA

with the Steel-Dwass test).

Fig. S14. Sema3A regulates synapse elimination through receptors located on climbing

fibers.

(A, B) Digital representation of confocal microscopic images showing immunoreactivity of

calbindin (blue, Purkinje cells), the vesicular glutamate transporter VGluT2 (red, climbing

fiber terminals), and enhanced green fluorescent protein (EGFP, green) in cerebellum tissue

with climbing fibers expressing scrambled PlxnA4 miRNA (PlxnA4-SCR) (A) or

microRNA against PlxnA4 (PlxnA4-KD) (B). White arrows indicate VGluT2 terminals that

contact Purkinje cells. (C) Frequency distribution of the number of perisomatic climbing

fiber terminals in Purkinje cells from PlxnA4-SCR (white columns, n = 38) and

PlxnA4-KD (green columns, n = 32) mice at postnatal days (P)14–P15. * P < 0.05

(Mann–Whitney U test).

Fig. S15. Knocking down PlxnA2 in climbing fibers has no effect on synapse

elimination.

(A) Examples of climbing fiber-mediated excitatory postsynaptic currents in Purkinje cells

associated with climbing fibers expressing microRNA against PlxnA2 (PlxnA2-KD) and by

cells from regions in which transfected climbing fibers were absent (Control). Scale bars,

0.5 nA and 5 ms. (B) Frequency distribution of the number of climbing fibers innervating

each control (white columns, n = 36) and PlxnA2-KD (red columns, n = 29) Purkinje cell at

postnatal day (P)12–P15. ns indicates no significant difference (Mann–Whitney U test).

Fig. S16. Rescue of the effect of PlxnC1- or ItgB1-knockdown by co-expression of

microRNA-resistant PlxnC1 or ItgB1, respectively.

(A, B) Examples of climbing fiber-mediated excitatory postsynaptic currents in Purkinje

cells associated with climbing fibers expressing a microRNA (miRNA)-resistant form of

PlxnC1 together with a miRNA against PlxnC1 (plxnC1-RES) (A), and with climbing

fibers expressing a miRNA-resistant form of ItgB1 together with a miRNA against ItgB1

(ItgB1-RES) (B), and in Purkinje cells sampled from regions in which transfected climbing

fibers were absent (Control) (A, B). Scale bars, 0.5 nA and 5 ms. (C, D) Frequency

distribution of the number of climbing fibers innervating each control (white columns, n =

30) and PlxnC1-RES (beige columns, n = 19) Purkinje cell at postnatal day (P)21–P30 (C),

and in control (n = 20) and ItgB1-RES (yellow columns, n = 19) (D) Purkinje cells. ns

indicates no significant difference (Mann–Whitney U test).

Fig. S17. Sema7A regulates synapse elimination through receptors located on climbing

fibers.

(A–D) Digital representation of confocal microscopic images showing immunoreactivity of

calbindin (blue, Purkinje cells), the vesicular glutamate transporter VGluT2 (red, climbing

fiber terminals), and enhanced green fluorescent protein (EGFP) in cerebellum tissue with

climbing fibers expressing scrambled PlxnC1 microRNA (miRNA) (PlxnC1-SCR) (A),

miRNA against PlxnC1 (PlxnC1-KD) (B), scrambled ItgB1 miRNA (ItgB1-SCR) (C), or

miRNA against ItgB1 (ItgB1-KD) (D). White arrows in (B) and (D) indicate VGluT2

signals on Purkinje cell somata with GFP-labeled climbing fibers. (E, F) Frequency

distribution of the number of perisomatic climbing fiber terminals in PlxnC1-SCR (white

columns, n = 38) and PlxnC1-KD (orange columns, n = 35) Purkinje cells during postnatal

day (P)21–P22 for (E), and for ItgB1-SCR (white columns, n = 36) and ItgB1-KD (yellow

columns, n = 33) cells (F). *P < 0.05 and **P < 0.01 (Mann–Whitney U test).

Fig. S18. Double knockdown of PlxnC1 and ItgB1 in climbing fibers impairs synapse

elimination to the same extent as a single knockdown of either molecule.

(A) Examples of climbing fiber-mediated excitatory postsynaptic currents in Purkinje cells

associated with climbing fibers expressing microRNAs against PlxnC1 and ItgB1

(PlxnC1/ItgB1-DKD), and in cells sampled from regions in which transfected climbing

fibers were absent (Control). Scale bars, 0.5 nA and 5 ms. (B) Frequency distribution of the

number of climbing fibers innervating each control (white columns, n = 30), PlxnC1-KD

(orange columns, n = 45), ItgB1-KD (yellow columns, n = 42), and PlxnC1/ItgB1-DKD

(beige columns, n = 30) Purkinje cell during postnatal day (P)21–P30. The data for

PlxnC1-KD and ItgB1-KD are taken from Figure 4D and 4F, respectively. *P < 0.05. ns

indicates no significant difference (Mann–Whitney U test).

Fig. S19. Impairment of climbing fiber synapse elimination by expression of

constitutively active cofilin in climbing fibers.

(A) Sample climbing fiber-mediated excitatory postsynaptic currents in Purkinje cells

associated with climbing fibers expressing constitutively active cofilin (CA-cofilin) and in

cells sampled from regions in which the transfected climbing fibers were absent (Control).

Scale bars, 0.5 nA and 5 ms. (B) Frequency distribution of the number of climbing fibers

innervating each Purkinje cell for the control (white columns, n = 49) and for CA-cofilin

(pink columns, n = 21) during postnatal day (P)21–P30. *P < 0.05 (Mann–Whitney U test).

Fig. S20. Impairment of climbing fiber synapse elimination by knockdown of focal

adhesion kinase (FAK) in climbing fibers.

(A) Knockdown efficacy of FAK. HEK 293T cells were transfected with the indicated

constructs and then double immunostained with an anti-GFP antibody and an anti-FLAG

antibody. Scale bar, 30 µm. (B) Fluorescence intensity of FAK relative to that of GFP in

control cells (white column), knockdown cells (purple column), and cells expressing a

microRNA (miRNA)-resistant form of cDNA (light purple column). ***P < 0.005

(Steel-Dwass test). (C) Example of climbing fiber-mediated excitatory postsynaptic

currents in Purkinje cells associated with climbing fibers expressing miRNA against FAK

(FAK-KD), a miRNA-resistant form of FAK together with a miRNA against FAK

(FAK-RES), and in Purkinje cells sampled from regions in which the transfected climbing

fibers were absent (Control). Scale bars, 0.5 nA and 5 ms. (D) Frequency distribution of the

number of climbing fibers innervating each control (white columns, n = 31), FAK-KD

(purple columns, n = 31), and FAK-RES (light purple columns, n = 31) Purkinje cell. *P <

0.05 (Mann–Whitney U test).

Fig. S21. Cofilin and focal adhesion kinase (FAK) mediate climbing fiber synapse

elimination downstream of PlxnC1 and ItgB1, respectively.

(A, C) Examples of climbing fiber-mediated excitatory postsynaptic currents in Purkinje

cells associated with climbing fibers expressing constitutively active cofilin together with a

microRNA (miRNA) against PlxnC1 (CA-cofilin/PlxnC1-KD), in cells sampled from

regions in which the transfected climbing fibers were absent (Control) (A). Examples of

climbing fiber-mediated excitatory postsynaptic currents in Purkinje cells associated with

climbing fibers expressing miRNAs against FAK and ItgB1 (FAK/ItgB1-DKD), and in

cells sampled from regions in which the transfected climbing fibers were absent (Control)

(C). Scale bars, 0.5 nA and 5 ms. (B, D) Frequency distribution of climbing fibers

innervating each control (white columns, n = 35), CA-cofilin (pink columns, n = 21), and

CA-cofilin/PlxnC1-KD (wine red columns, n = 20) Purkinje cell (B), and each control (n =

37), FAK-KD (nile blue columns, n = 31), and FAK/ItgB1-DKD (iron blue columns, n =

22) Purkinje cell (D) during postnatal day (P)19–P30. The data for CA-cofilin and

FAK-KD presented in Figure S19B and S20D are also used here. *P < 0.05. ns indicates no

significant differences (Mann–Whitney U test).

Fig. S22. Summary of the effects of Sema3A and Sema7A on climbing fiber synapse

elimination.

Previous studies demonstrate that the early phase requires P/Q-type voltage-gated Ca2+

channels (P/Q-VGCC) and GABAergic inhibition (2). The late phase depends on

GABAergic inhibition (and (presumably) also on P/Q-VGCC) (8), and on neural activity

along mossy fibers-to-parallel fibers, which drives metabotropic glutamate receptor 1

(mGluR1) signaling in Purkinje cells (2). The present results suggest that

Sema3A-to-PlxnA4 signaling maintains the synapses of weaker climbing fibers and

strengthens/maintains the synapses of the strongest climbing fibers during both the early

and late phases of climbing fiber elimination. By contrast, Sema7A-to-PlxnC1/ItgB1

signaling facilitates selective elimination of synapses from weaker climbing fibers during

the late phase of climbing fiber elimination. This process is controlled by mGluR1

signaling, which is activated by neural activity. The model presented here shows that

elimination of the weaker climbing fibers maintained by Sema3A-to-PlxnA4 signaling

depends on P/Q-VGCC, GABAergic inhibition, and mGluR1-to-Sema7A-to-PlxnC1/ItgB1

signaling.

Table S1. Expression of mRNAs for Sema3A and Sema7A

Levels of Sema3A (A) and Sema7A (B) mRNA expression in the cerebellum during

postnatal development from postnatal day (P)4–P18. Data are derived from the DNA

microarray analysis shown in Fig. S2. The expression levels of Sema3A and Sema7A were

normalized to that of an internal standard: glyceraldehyde-3-phosphate dehydrogenase

(GAPDH, top) or -actin (ACTB, bottom).

Table S2. Kinetics of CF-EPSCs for Sema3A-knockdown and PlxnA4-knockdown.

(A) Total amplitudes and disparity ratios for climbing fiber-mediated excitatory

postsynaptic currents (CF-EPSCs) in individual Sema3A-knockdown (postnatal day

(P)11–15 and P21–P30) and PlxnA4-knockdown (P11–15) Purkinje cells in vivo. The total

amplitude is lower in Sema3A-knockdown and PlxnA4-knockdown Purkinje cells than in

control Purkinje cells. There was no difference in the disparity ratio between control and

knockdown Purkinje cells. Disparity ratio = (A1/AN+ A2/AN+…..+ AN-1/AN)/(N-1); (N > 1).

Ai is the EPSC amplitude for the climbing fiber i, and N is the number of climbing fibers

for a given Purkinje cell. (B) Kinetics and paired-pulse ratio of the CF-EPSCs from

Sema3A-knockdown and PlxnA4-knockdown Purkinje cells in vivo. CF-EPSCs were

divided into three groups: CF-EPSCs from mono-innervated Purkinje cells (CF-mono), the

largest CF-EPSCs from individual multiply-innervated Purkinje cells (CF-multi-S), and

other (smaller) CF-EPSCs from individual multiply-innervated Purkinje cells (CF-multi-W).

Amplitudes are measured at a holding potential of -20 mV. Data are expressed as the mean

± SEM. P-values were determined by the Mann–Whitney U test (compared with values for

the corresponding controls). *P < 0.05 and **P < 0.01.

Table S3. Kinetics of CF-EPSCs for Sema7A-knockdown, PlxnC1-knockdown, and

ItgB1-knockdown.

(A) Total amplitudes and disparity ratios for climbing fiber-mediated excitatory

postsynaptic currents (CF-EPSCs) in individual Purkinje cells (holding potential, -20 mV)

for Sema7A-knockdown, PlxnC1-knockdown, and ItgB1-knockdown in vivo. There was no

difference in the total amplitude and disparity ratio between control and knockdown

Purkinje cells. (B) Kinetics and paired-pulse ratios of CF-EPSCs from Sema7A-knockdown,

PlxnC1-knockdown, and ItgB1-knockdown Purkinje cells in vivo. P-values were

determined by the Mann–Whitney U test (compared with values for the corresponding

controls). Amplitudes were measured at a holding potential of -20 mV. Data are expressed

as the mean ± SEM.

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