Protein, Nestin

This study was carried out in strict accordance with the recommendations in the
Guide for the Care and Use of Laboratory Animals of the National Institutes of
Health. All procedures used were approved by the University of Texas
Southwestern Medical Center Institutional Animal Care and Use Committee APN
2007-0065. All efforts were made to minimize suffering.
Ascl1^CreERT2 knock-in mice were generated by
replacing the Ascl1 coding region with
CreER^T2[8] (link) and
Frt-Neo-Frt cassettes. The targeting strategy was the same used to generate
Ascl1^GFP knock-in mice [9] (link). The endogenous ATG was
replaced by a short sequence containing a PacI site and a consensus Kozak site.
The correct targeting event was identified by Southern analysis of EcoRI
digested DNA using 5′ and 3′ probes. After obtaining germ line
transmission in the Ascl1^{CreERT2-Frt-Neo-Frt} mice,
they were crossed with FLPe mice [10] (link) to remove the neomycin
cassette resulting in Ascl1^CreERT2 mice.
For PCR genotyping, the following primers were used: 5′-AAC TTT CCT CCG GGG CTC GTT
TC-3′ (Sense Ascl1 5′UTR) and 5′-CGC CTG GCG ATC CCT GAA CAT
G-3′ (Anti sense Cre) giving a PCR product of 247 bp.
Tamoxifen (TAM) induction of Cre recombinase was accomplished by intraperitoneal
injection of
Ascl1^CreERT2/+;R26R^YFP/YFPpostnatal day 50 (P50) mice with 180 mg/kg/day TAM (Sigma, T55648) in sunflower
oil on five consecutive days. Brains were harvested at the times specified after
TAM and processed as described [7] (link), [11] (link). R26R^YFP and
Nestin::GFP mice have been previously described [12] (link), [13] (link).
For immunofluorescence staining, free floating sections or sections mounted on
slides were incubated in the appropriate dilution of primary antibody in
PBS/3% donkey (or goat) serum/0.2% NP-40 (or 0.2% Triton
X-100), followed by appropriate secondary antibody conjugated with AlexaFluor
488, 568, or 594 (Molecular Probes). Mouse monoclonal antibodies used were:
Ascl1 (1∶750, RDI Fitzgerald, 10R-M106B), NeuN (1∶1000, Chemicon,
MAB377), GFAP (1∶400, Sigma, G3893). Rabbit polyclonal antibodies used
were: GFP (1∶500, Molecular Probes, A6455), GFAP (1∶500, DAKO,
Z0334), Ki67 (1∶500, Neomarker), Sox2 (1∶2000, Millipore). Goat
polyclonal antibodies used were: DCX (1∶200, Santa Cruz) and NeuroD1
(1∶200, Santa Cruz). Chick GFP (1∶500, Aves Lab) was also used.
Confocal imaging was carried out on a Zeiss LSM510 confocal microscope.
Ascl1⁺ fluorescence intensity levels were classified as high
or low using ImageJ and setting a threshold of pixel intensity for
Ascl1^Low (314–599 units) and Ascl1^High (>600
units). For cell number counts, three Nestin::GFP mice were
analyzed to place Ascl1⁺ progenitors in the adult neural stem
cell lineage. For in vivo genetic tracing experiments using the
Ascl1^CreERT2 knock-in line, at least two
Ascl1^CreERT2/+;R26R^YFP/YFPmice per each harvest time point (7, 30, or 180 days post-TAM) were used. For
co-localization data with each stage-specific marker, 150–500
YFP⁺ cells per animal were counted.

Mouse E9.5 embryos were partially digested in warm PBS containing 5 mg/ml pancreatin (SIGMA) for 3 min, followed by isolation of caudal neural tubes using fine forceps in ice-cold PBS. The isolated neural tubes was passed through a 27-gauge needle (TERUMO, Tokyo, Japan) ten times to dissociate cells, which were subsequently cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s medium (GIBCO) and F-12 nutrient (GIBCO) containing 0.6% glucose, 5 mM HEPES buffer, 25 μg/ml insulin, 100 μg/ml transferrin, 20 nM progesterone, 60 μM putrescine, 30 nM selenium chloride, 10 ng/ml FGF-2, and 20 ng/ml EGF, as described by Tropepe et al.^{45 (link)}. Dissociated cells were seeded onto a collagen type I (Nippi, Tokyo, Japan) coated 96-well cell culture plate (3596, Corning, NY) and cultured for 24 h at 37 °C in a humidified atmosphere containing 5% CO₂ and 3% O₂. Cells were subsequently fixed with 4% PFA and permeabilised with 0.1% TritonX-100, followed by blocking with PBS containing 10% FBS for 1 h at room temperature. Cells were then incubated overnight with the primary antibody at 4 °C, excess of which was removed by three washes with PBS, prior to treatment with Alexa labelled secondary antibodies. Subsequent to three washes with PBS, immunostained cells were visualized using the high-throughput high-content imaging system Opera Phenix (Perkin Elmer, Waltham, MA), and data were analysed using Harmony 4.5 (Perkin Elmer). On average 342 ± 99 cells were selected as the Nestin-positive cells in each well and mean fluorescent intensity of Alexa in the selected cells was calculated.

All studies were approved by the Service de la consommation et des affaires vétérinaires of Geneva in Switzerland. Mice were group-housed with littermates in standard housing on a 12:12-hour light/dark cycle. Nestin-GFP reporter mice (66 (link)) have been obtained from S. Jessberger (University of Zürich), hGFAP-CreERT2 mice (34 (link)) from N. Toni (University Hospital Lausanne, CHUV), Mpc1 fl/fl mice (33 (link)) from E. Taylor (University of Iowa), and tdTom fl-STOP-fl [Ai14 (35 (link))] from I. Rodriguey (University of Geneva). All mice were on a C57BL/6 background. Genotyping was performed on DNA extracted from phalange biopsies using the following primers: hGFAP-CreERT2: F-CAG GTT GGA GAG GAG ACG CAT CA, R-CGT TGC ATC GAC CGG TAA TGC AGG C; MPC1 fl/fl: F1-CCT ATT CTC TAG AAA GTA TAG GAA CTT CGT CGA, F2-GTG AGC CCA GAG CTA CGA AGG ATC GGC, F3-GGA AAG AAA AAG GTG TCC AAT TTT AGC TCT GCA; tdTom fl STOP-fl: F 5′-CTG TTC CTG TAC GGC ATG G-3′, R 5′-GGC ATT AAA GCA GCG TAT CC-3′, tdTom WT/WT:-F 5′-AAG GGA GCT GCA GTG GAG TA-3′, R 5′-CCG AAA ATC TGT GGG AAG TC-3′.

All images were collected on a Leica confocal imaging system (TCS SP8) with a 20× or 63× oil immersion objective, Leica Thunder imaging system (DMi8) with a 20× objective, and Spinning Disk confocal imaging system (Nikon Ti2/Yokogawa CSU-W1) with 60× and 100× oil immersion objectives. For the quantification of recombined tdTom-positive cells with specific markers, serial sections starting from the beginning of the DG were used. Quantification of tdTom-positive and Ki67-positive cell labeling with different markers, DCX and NeuN, was performed using the software Fiji (ImageJ 2.0.0). To confirm Ki67, DCX, and NeuN colocalization with tdTom, single optical sections at 63× magnification were used.
For MPC1 quantification in tissue, z-stacks of single Nestin-GFP cells positive or negative for Ki67 (63× objective) were acquired. Quantification of MPC1 and GFP signal was performed using IMARIS software (Bitplane 9.9.1). First, the GFP volume reconstruction was performed using surface plugin. To confirm the signal of MPC1 in GFP-expressing cells, MPC1 signal was masked in GFP-positive cells using mask plugin. Last, MPC1 volume reconstruction of masked signal was performed using surface plugin. Eighteen to 26 GFP-positive cells were analyzed per group.
For Sholl analysis, the Cre virus–transfected GFP and tdTom double-positive newborn neurons were imaged with a 63× [0.75 numerical aperture (NA)] objective. Z-stacks were taken at 1-μm intervals, and dendrites were traced using the Neurolucida software (version 10, mbs Bioscience). Fifteen to 18 neurons from four mice per group were analyzed. Dendritic spine density and spine morphology were assessed as previously described (69 , 70 (link)) with little adjustments. Briefly, dendrites of 20 to 30 Cre virus–transfected GFP and tdTom double-positive newborn neurons were imaged using a 63× (2.5 NA) objective. The dendritic length and the number of spines were analyzed using the software Fiji (ImageJ 2.0.0). Spine density was expressed as the number of the spines divided by dendritic length. Spine morphology was classified in two groups on the basis of the maximal diameter of the spine head, as measured on maximal projections with Fiji (ImageJ 2.0.0). Immature spines (thin spines) were defined as 0.25 to 0.6 μm and mature spines (mushroom) >0.6 μm. The percentage of each type of dendritic spine was then expressed by neuron for each mouse (20 to 30 neurons per mouse, four mice per group). The data were expressed as the ratio between immature spines and mature spines. All images were analyzed in a blinded manner.