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
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
2
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
3
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
4
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
8
(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
9
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)