1

Supplementary Information

Dll1 maintains quiescence of adult neural stem cells and segregates

asymmetrically during mitosis

Daichi Kawaguchi, Shohei Furutachi, Hiroki Kawai, Katsuto Hozumi and

Yukiko Gotoh

SVZ (dorsal) SVZ (ventral)

Dll1

imm

unos

tain

ing

Dll1

FIS

H H

oech

st

Dll1 immunostainingDll1 FISHHoechst

Dll1 immunostainingEGFR immunostainingHoechst

a b c

d

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Supplementary Figure S1. Dll1-immunopositive puncta are detected in most Dll1-mRNA

expressing cells in the adult mouse SVZ. (a-d) Dll1 protein and Dll1 mRNA were detected

simultaneously in the adult mouse SVZ ((a) dorsal SVZ, (b) ventral SVZ).

Dll1-immunopositive puncta were detected using Dll1-antibody, and Dll1-mRNA was

detected by fluorescence in situ hybridization (FISH). (c, d) The boxed region in (b) is shown

at higher magnification. (d) EGFR-immunostaining was used as a counter-staining to show

that both the Dll1-immunopositive puncta and the Dll1-mRNA belong to the same cell. Note

that 71.7 ± 1.1% of Dll1-mRNA expressing cells were positive for Dll1-immunopositive

puncta (total counted cells = 169 cells, n = 3 brains). Scale bars, 5 µm.

-24 min 4 min0 min 140 min 204 min-40 min-64 min

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Supplementary Figure S2. Low magnification images of embryonic neocortical slice

culture. Arrows and arrowheads indicate a Dll1-EGFP+ dividing pair shown in Fig. 6a and

Supplementary Movie 4. Dotted lines indicate the ventricular surface. Scale bar, 10 μm.

Dll1-EGFP H2B-mCherry

H2B-mCherrySox2Tbr2

Fig.

6b,

Sis

ter (

i)

H2B-mCherryH2B-mCherrySox2H2B-mCherry

Fig.

6b,

Sis

ter (

ii)

Fig. 6bSister (i)

Fig. 6bSister (ii)

H2B-mCherrySox2Tbr2

a bSox2 Tbr2

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Supplementary Figure S3. The non–Dll1-inheriting daughter cell maintains the

undifferentiated NSC state after asymmetric NSC division. (a) The

immunohistofluorescence staining of the cortical slice after time-lapse imaging is shown. The

cortical slice was subjected to immunofluorescence staining of Sox2 and Tbr2. Sister (i) and

(ii) denote the same sister cells indicated in Fig. 6b and Supplementary Movie 5. Dotted lines

indicate the ventricular surface. Cell position of the non–Dll1-inheriting daughter cell (sister

(ii)) is in the ventricular zone. (b) High magnification images of sister (i) and (ii) shown in (a).

The undifferentiated state of the non–Dll1-inheriting daughter cell (sister (ii)) was confirmed

as Sox2+Tbr2–. Scale bars, 10 µm.

Dll1p-Vim Aurora BHoechst

GFAPp-Vim Aurora BHoechstDll1 Hoechst p-Vim Aurora B Hoechst GFAP Hoechst

5

Supplementary Figure S4. The non–Dll1-inheriting daughter cell expresses GFAP in the

adult SVZ. Dividing daughters of an activated NSC (GFAP+ division) in the SVZ of the adult

brain. Sections were stained for Dll1, phospho-vimentin, Aurora B, and GFAP, as indicated.

Scale bar, 5 µm.

6

Supplementary Methods

Expression constructs and antibodies. The plasmids pcDNA5/TO-H2B-mCherry,

Fucci probe mCherry-hGeminin(1/60), vesicular stomatitis virus G (VSV-G) expressing

vector, and pMXs vector were kindly provided by Y. Watanabe, A. Miyawaki, H. Song,

and T. Kitamura respectively. Dll1 cDNA was cloned from mouse brain extract by

reverse transcription and the polymerase chain reaction and was verified by DNA

sequencing. For construction of an expression vector for Dll1 tagged at its

COOH-terminus with EGFP, the entire Dll1 coding sequence without the stop codon

was cloned from the mouse Dll1 cDNA by the polymerase chain reaction, verified by

DNA sequencing, and then inserted into pEGFP-N1 (Clontech) to yield

pEGFP-N1-Dll1. For retrovirus induction, Dll1-EGFP and mCherry-hGeminin(1/60)

were inserted to the pMXs vector to yield pMXs-Dll1-EGFP and

pMXs-mCherry-hGeminin(1/60).

Antibodies used for immunostaining included mouse monoclonal antibodies

to bromodeoxyuridine (for IdU, BD Bioscience) at a 1:500 dilution, to βIII-tubulin

(TuJ1, Babco) at 1:1000, to GFAP (clone GA5, Millipore) at 1:100, to Ascl1 (BD

Pharmingen) at 1:100, to phospho-vimentin (4A4, MBL) at 1:500, to Aurora B (BD

Transduction Laboratories) at 1:100, to S100β (SH-B4, Sigma) at 1:50, and to Sox2

(R&D Systems) at 1:200; a rat monoclonal antibody to bromodeoxyuridine (for CldU,

Abcam) at 1:200; rabbit monoclonal antibodies to cleaved Notch1 (NICD) (D3B8, Cell

Signaling Technology) at 1:100 and to EGFR (E114, Epitomics) at 1:100; rabbit

polyclonal antibodies to GFAP (Dako) at 1:1000, to Ki67 (Novocastra) at 1:100, to

Aurora B (Cell Signaling Technology) at 1:100, and to Tbr2 (Abcam) at 1:1000; sheep

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polyclonal antibodies to Dll1 (R&D Systems) at 1:100 and to EGFR (Upstate

Biotechnology) at 1:100; and goat polyclonal antibodies to Dcx (Santa Cruz

Biotechnology) at 1:500. Alexa Fluor–labeled secondary antibodies and Hoechst 33342

(for nuclear staining) were obtained from Molecular Probes. Sox2 was used in order to

distinguish between adult NSCs (GFAP+Sox2+) and niche astrocytes (GFAP+Sox2–) in

the SVZ13. A Zenon labeling kit (Molecular Probes) was used to label mouse antibodies

when two mouse antibodies were used simultaneously.

In utero electroporation and time-lapse imaging. Procedures were performed as

described previously61-63, with minor modifications. In brief, E13.5 ICR embryos (E1

was defined as 12 h after detection of the vaginal plug) were subjected to

electroporation with the plasmids pEGFP-N1-Dll1 (1.5 µg/µl) and

pcDNA5/TO-H2B-mCherry (0.2 µg/µl) with the use of a CUY21E instrument (Tokiwa

Science). The uterine horn was then returned to the abdominal cavity, and the embryos

were allowed to continue development for 15-19 h in utero. The embryos were then

harvested, and cortical slices were prepared and cultured in collagen gels with enriched

culture medium (DMEM/F-12 medium (Invitrogen) supplemented with B27 and N2

supplements (Invitrogen), basic fibroblast growth factor (10 ng/ml), epidermal growth

factor (10 ng/ml), 5% horse serum, and 5% fetal bovine serum). Time-lapse images

were obtained with a confocal microscope (Leica TCS-SP5) equipped with a 63×/1.20

water objective lens (HCX Plan Apo CS, Leica), a motorized XYZ stage (Leica), and a

controlled stage-top incubator (40% O2, 5% CO2; Tokai hit). Images were obtained at

multiple positions and at intervals of 3-7 min. z-Series images were reconstructed and

analyzed with LAS AF software (Leica). Images were processed with Photoshop CS

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(Adobe), QuickTime Pro (Apple) and Image J (US National Institutes of Health)

software. For determination of the fate of daughter cells of NSC divisions, cortical

slices in collagen gels were fixed immediately after time-lapse imaging with 4%

paraformaldehyde at 4°C for 1-6 h and were then subjected to immunohistofluorescence

analysis according to the procedure described above for whole-mount preparations. The

neuronal progenitor marker Tbr2 was previously found to be expressed asymmetrically

in one of the daughter cells in most Tbr2-expressing daughter pairs, with the onset of

expression being ~4 to 6 h after NSC division63. To monitor asymmetric cell fate on the

basis of Tbr2 expression, we therefore examined the fate of daughter cells at ~6 to 12 h

after NSC division.

Primary adult SVZ culture. Procedures were performed as described previously64,65,

with minor modifications. In brief, SVZs were dissected from the lateral wall of the

lateral ventricle of adult (2 months) ICR mice in DMEM/F12 (Sigma) and then

dissociated using the Nerve-Cell Culture System/Dissociation Solution (Sumitomo

Bakelite). Dissociated cells were cultured on poly-D-lysine (Sigma) coated dishes (glass

base dishes (Iwaki) for time-lapse imaging) at a density of 300-500 cells per mm2 in

DMEM/F12 Glutamax (Gibco) supplemented with 40 ng/ml FGF2 (Peprotech), B-27

Supplement (Gibco), 2mM L-Glutamine (Gibco), 100 units/ml penicillin (Invitrogen),

100 mg/ml streptomycin (Invitrogen), buffered with 8mM Hepes (Gibco). For

immunocytofluorescence analysis, cells were cultured for 3 days, and then subjected to

immunocytofluorescence analysis. For time-lapse imaging, cells were mixed with

recombinant retroviruses expressing Dll1-EGFP and mCherry-hGeminin(1/60) for

16-20 h, then incubated with fresh medium for 18-30 h. Recombinant retroviruses were

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produced in Plat-GP cells (kindly provided by T. Kitamura and H. Song) transfected

with pMXs vector and VSV-G expression vector. Time-lapse images were acquired

with using a Cell Voyager CV1000 (Yokogawa). Images were obtained at intervals of 7

min. They were processed with Cell Voyager CV1000 Measurement Software

(Yokogawa), Photoshop CS (Adobe), QuickTime Pro (Apple) and Image J (US

National Institutes of Health) software. After time-lapse imaging, the cultured cells

were subjected to immunocytofluorescence staining to determine the fate of daughter

cells.

Supplementary References

61. Konno, D. et al. Neuroepithelial progenitors undergo LGN-dependent planar

divisions to maintain self-renewability during mammalian neurogenesis. Nat. Cell

Biol. 10, 93-101 (2008).

62. Miyata, T. & Ogawa, M. Twisting of neocortical progenitor cells underlies a

spring-like mechanism for daughter-cell migration. Curr. Biol. 17, 146-151 (2007).

63. Ochiai, W. et al. Periventricular notch activation and asymmetric Ngn2 and Tbr2

expression in pair-generated neocortical daughter cells. Mol. Cell. Neurosci. 40,

225-233 (2009).

64. Costa M. R. et al. Continuous live imaging of adult neural stem cell division and

lineage progression in vitro. Development 138, 1057-1068 (2011).

65. Mirzadeh Z., Doetsch F., Sawamoto K., Wichterle H., Alvarez-Buylla A. The

subventricular zone en-face: wholemount staining and ependymal flow. J. Vis. Exp.

39, 1938 (2010)