www.sciencemag.org/cgi/content/full/science.1244505/DC1

Supplementary Materials for

BTBD3 Controls Dendrite Orientation Toward Active Axons in Mammalian Neocortex

Asuka Matsui, May Tran, Aya C. Yoshida, Satomi S. Kikuchi, Mami U, Masaharu Ogawa, Tomomi Shimogori*

*To whom correspondence should be addressed. E-mail: tshimogori@brain.riken.jp

Published 31 October 2013 on Science Express

DOI: 10.1126/science.1244505

This PDF file includes:

Material and Methods Figs. S1 and S2 References

Supplemental section Supplementary material Fig. S1

Mouse somatosensory barrel development in early postnatal stage.

Thalamocortical axons and cortical neurons were stained by anti-vGlut2 and

anti-RORc antibodies, respectively. Coronal section (A-D) and tangential section

(E-H) of the cortex is shown at different developmental time points. Scale bar in

A is 100 µm.

Fig. S2

(A) Btbd3 knockdown in mice by shRNA construct. Btbd3 shRNA is

electroporated at E13.5 and Btbd3 expression was assessed by in situ

hybridization at P6, resulting in an approximately 70% reduction in gene

expression compare to control brain. Scale bar is 200 µm.

(B) Control YFP plasmid (mock), human BTBD3 or human BTBD3 and shRNA

construct was electroporated at E13.5 and brains were harvested at E16.5.

Expression of BTBD3 mRNA was tested with using a human BTBD3-specific

primer. (C) Morphological changes of primary dendrites during barrel formation.

Electroporation was performed at E13.5 to transfect the YFP construct. Brains

were harvested at P0, P2, P4 and P6, tangentially sectioned and stained with

anti-GFP antibody to reveal dendritic morphology. (D) Number of primary

dendrites was counted using the Sholl method. * p < 0.05, ** p < 0.01, t test.

Materials and Methods. Animals Outbred ICR (CD-1) timed pregnant mice were obtained from Japan SLC.

Midday of the day of vaginal plug discovery is considered embryonic (E) day 0.5.

NR1flox/flox mice were crossed with Emx1 cre mice to obtain conditional KO (10).

Early postnatal mice were anesthetized with a lethal dose of pentobarbitone

(100 mg/kg), and after three failed attempts to elicit a foot withdrawal reflex, the

animals were transcardially perfused with 4% paraformaldehyde in

phosphate-buffered saline. Normally pigmented, sable ferrets, Mustela putorius

furo, were purchased from Marshall Farms (North Rose, NY). All procedures

were performed in accordance with a protocol approved by RIKEN Institutional

Animal Care.

In utero electroporation Mouse in utero electroporation and ferret electroporation were performed as

described previously (18, 28) with combination of sparse labeling method

provided high resolution of dendritic morphology (29). For targeting mouse

layer IV neurons, electroporation was performed at embryonic day (E) 13.5.

For targeting ferret layer IV neurons, electroporation was performed at E34.

In situ hybridization Brains were removed, fixed overnight in 30% sucrose/4% paraformaldehyde, and

sectioned in the coronal plane on a Leica sledge microtome at 28µm. In situ

hybridization was performed as described previously (30).

Immunohistochemistry Anti-RORc (H3925, Perseus Proteomics, Tokyo, Japan) and anti-vGlut2 (135 404,

Synaptic Systems, 1/400) primary antibodies were detected with the following

secondary antibodies: horse anti-mouse IgG (Vector, FI-2000) and

Cy3-conjugated goat anti-rabbit IgG (Chemicon, AP132C).

Western blotting and immunocytochemistry Whole brain was collected after quick decapitation and washed in cold PBS to

remove any contaminating blood. Somatosensory cortex was immediately

dissected and kept in cold RIPA buffer [20 mM Tris, pH 7.4, 150mM NaCl, 2mM

EDTA, 1% NP-40, 1% Na deoxycholate, 0.1% SDS and protease inhibitor

cocktail (04080, Nacalai tesque, Japan)]. To collect the cytoplasmic fraction,

somatosensory cortex was kept in cold extract buffer and centrifuged [20mM

HEPES, pH7.6, 20% Glycerol, 10mM NaCl, 1.5mM MgCl2, 0.2mM EDTA, pH 8.0,

1mM DTT, 0.1% NP-40 and protease inhibitor cocktail (Nacalai tesque)]. After

collecting supernatant of cytoplasmic extraction, the pellet is homogenized with

cold extract buffer to extract nuclear fraction [20mM HEPES, 20% Glycerol,

500mM NaCl, 1.5mM MgCl2, 0.2mM EDTA, 0.1% Glycerol and protease

inhibitor cocktail]. Primary antibodies and concentration for western blotting were,

rabbit anti-BTBD3 antibody (HPA042048; ATLAS, 1:500), mouse anti-α-tubulin

antibody (T6199; SIGMA-ALDRICH, 1:20,000), mouse anti-c-Raf antibody

(610151; BD Transduction Laboratories, 1:2,000), rabbit anti-MEF2D antibody

(#AB2263; Millipore, 1:2,000). For secondary antibodies, we used

peroxidase-conjugated goat anti-rabbit (# 7074S, Cell Signaling), anti-mouse

IgG antibody (#7076S, Cell Signaling, 1:10,000). The signals were visualized

using chemiluminescent detection reagents (02230; Chemi-Lumi One Super,

Nacalai tesque). Images of the signals were captured with LAS-3000UV mini

(Fujifilm).

DNA Constructs and shRNA Full length Btbd3 (FANTOM clone, D430043L03) was subcloned into Tol2

plasmid vector and electroporated with T2TP vector for permanent transfection

(31). Small hairpin (sh) RNA plasmid was purchased from Santa Cruz (BTBD3

shRNA Plasmid (m): sc-141776-SH). Btbd3-HA construct is generated using

PCR to add HA tag on C-terminus of mouse Btbd3.

QT-PCR Total RNA extractions were carried out with Trizol reagent (Invitrogen,

15596-026) according to the manufacturer's instructions. RNA obtained from

brains was reverse transcribed by using SuperScript III First-Strand Synthesis

System (18080-051, Invitrogen). Synthesized cDNA was used for PCR reaction

with using following primers.

Ferret Btbd3-F: 5’-TGAAATTGACTTGGCTGCTG-3’

Ferret Btbd3-R: 5’-GCTGCCTCAAAAACCACAAT-3’

Ferret b-actin-F: 5’-GGCATCCTGACCCTGAAGTA-3’

Ferret b-actin-R: 5’-CTTGATGTCACGCACGATTT-3’

Human Btbd3-F: 5’-TGTGTTCCATGCGATGTTTT-3’

Human Btbd3-R: 5’-AGGAGCACACAGGCATTCTT-3’

mouse b-actin-F: 5'-GACTTTGTACATTGTTTTG-3'

mouse b-actin-R: 5'-TGCACTTTTATTGGTCTCA-3'

Cell culture and pharmacological treatment Neuro2a cells were cultured in DMEM medium (# 08458, Nacalai tesque)

supplemented with 10% fetal bovine serum, high-glucose and pyrubic acid). The

cells were seeded in a 2 well chamber slide (177380, Thermo Scientific, NY,

USA) for 24hr and transfected with plasmid vectors by X-tremeGENE HP DNA

transfection Reagent (06 366 126 001, Roche). After transfection, cells were

cultured for 24hr and cell differentiation was initiated by serum starvation in

DMEM. After 24hr, to depolarize the cultured cells, 60mM KCL solution was

added to the culture medium. After a 1hr stimulation, cells were fixed in 4% PFA.

Cells were treated with 0.1% triton-X in PBS and blocked with 5% goat serum

solution in PBS. The cultures were incubated with rabbit anti-HA polyclonal

antibody (RBP-101P, CONVANCE, 1: 200). Primary antibody was detected by

incubation with Alexa 488-conjugated anti-rabbit IgG (A-21244, Invitrogen) at 1:

200.

Confocal microscopy 40 µm tangential mouse brain sections or coronal ferret brain sections were

immunostained with anti-GFP and imaged using confocal microscopy.

Illustrations Contrast, brightness, and sharpness of entire images were adjusted by using

Adobe Photoshop CS5.

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