Histology and Immunohistochemistry (IHC)
The protocols for histology and IHC were the same as described previously (Zhang et al., 2007
; Tang et al., 2009
). Briefly, mouse embryos, from E10.5 to E12.5, were fixed with 1% PFA in PBS (4% PFA for Nkx6.1 antibody). Mice at embryonic day 18.5 (E18.5), postnatal day 0 (P0) and P21 were perfused and fixed with 4% PFA, and cryoprotected in 15%–30% sucrose solution and sectioned in the coronal plane using a Leica cryostat. Mouse brains were sectioned at 14 µm thickness (for stereology counting, brains were cut at 40 µm), and mounted on Superfrost glass slides.
Sections were incubated with primary antibody overnight and secondary antibodies for 1 hour, followed by incubation in DAB solution to detect signals. The primary antibodies in this study included: anti-BrdU antibody (1:500, MAB3222, Chemicon), anti-Foxa2 (1:20, 4C7, Developmental Hybridoma Study Bank [DHSB]), anti-Ki67 (1:200, RM9106-S0, Fisher), anti-Lmx1a (1:1,000, Gift of Dr. Mike German, UCSF), anti-Ngn2 (1:10, Gift of Dr. David Anderson, Caltech), anti-Pitx3 (1:300, Gift of Dr. Marten Smidt, University Medical Center Utrecht, the Netherlands), anti-Nkx2.2 (1:50, 74.5A5, DHSB), anti-Nkx6.1 (1:1,000, Gift of Dr. Mike German, UCSF), anti-Nurr1 (1:500, sc-990, Santa Cruz Biotechnology), anti-Otx2 (1:200, ab21990, Abcam), anti-phospho-histone H 3 (1:200, 06-570, Millipore), anti-Shh (1:200, #2207, Cell Signaling), anti-TuJ1 (1:2,000, MMS435P, Covance), anti-tyrosine hydroxylase (1:1,000, AB157, Chemicon), anti-tyrosine hydroxylase (1:500, ab113, Abcam), and anti-β-catenin (1:200, #9587, Cell Signaling). For stereology counting, sections were incubated for 1 hour with biotinylated IgG and avidin-biotin complex (Vector Laboratories, Burlingame, CA). Images were captured using a Nikon Eclipse E800 fluorescent microscope (Nikon, Japan), connected to a SPOT RT camera (Diagnostic Instruments Inc., Sterling Heights, MI), or a BX41 Olympus microscope equipped with Olympus DP70 CCD camera (Olympus, Japan). Images were captured using Spot Advance or Olympus DP Controller software programs or using a LSM 510 confocal microscope (Carl Zeiss 510 Microimaging).
BrdU Labeling of Dopaminergic Progenitors
We carried out 2 injection schemes. In the first scheme, the pregnant mice were injected with BrdU (50 µg/gm)(BD Bioscience) at E10.5 and E12.5, respectively, and sacrificed 2 hours later. In the second scheme, the pregnant mice were injected with BrdU at E10.5 and E11.5, respectively, and sacrificed 24 hours later (Zhang et al., 2007
; Tang et al., 2009
In Situ Hybridization
hybrydization were the same as described previously (Zhang and Huang, 2006
). Briefly, RNA probes for in situ
hybridization were prepared using plasmid cDNA clones for Shh
, cyclin D1
transcribed with T7 or T3 polymerase using digoxigenin (DIG)-labeling reagents and a DIG RNA labeling kit (Roche). Embryos were fixed overnight at room temperature in 4% PFA in DEPC-treated PBS, cryoprotected in 15% and 30% sucrose in DEPC PBS, and embedded in OCT. Sections were processed at 14µm. During hybridization, sections were first postfixed with 4% PFA, washed with acetylation solution and 1% Triton X-100. Then sections were prehybridized with hybridization buffer (Amresco) for 2–4 hrs before applying hybridization buffer containing DIG-labeled riboprobes (200–400 ng/ml) at 55°C overnight. The second day, slides were washed with 4xSSC, followed by RNase A (20µg/ml) treatment at 37°C for 45min, and subsequent washes with 2xSSC, 1xSSC and 0.5xSSC at room temperature. For visualizing the in situ
hybridization results, we used DIG Nucleic Acid Detection Kit (Roche). Finally, the slides were dried under room temperature and mounted with Crystal Mount (Biomeda).
The total number of TH+ neurons in SNpc and VTA was determined using the optical fractionator, an unbiased cell counting method not affected by the volume of reference (i.e. SNpc or VTA) or the size of the counted elements (i.e. neurons)(Zhang et al., 2007
; Tang et al., 2009
). Neuronal counts were performed using a computer-assisted image analysis system consisting of an Olympus BX-51 microscope equipped with a XYZ computer-controlled motorized stage and the StereoInvestigator software (Microbrightfield, Villiston, VT). TH+ neurons were counted in SNpc or VTA of every third section throughout the entire midbrain. Each section was viewed at lower power (4x) and outlined. At a random start, the numbers of TH-stained cells were counted at high power (60x oil; NA 1.4) using a 50×50 µm counting frame.
Ventral Midbrain (vMB) DA Progenitor Cultures
Primary cultures for dopamine neurons were prepared from ventral midbrain (vMB) using microisland methods according to published procedures (Takeshima et al., 1996
). Briefly, mouse embryos were collected from time-pregnant CD-1 (for E10.5 and E13.5 wild type
cultures) or Shh-Cre
(for E12.5 Shh-Cre
cultures) females. The ventral midbrain was dissected, dissociated after treatment with trypsin and cultured on coverslip coated with poly-D-ornithine (Sigma) and laminin (Sigma) at the density of 1.2x106
/ml. The dissociated cells were maintained in the DMEM-F12 (1:1) medium containing 10% FBS overnight. Then, the differentiated neurons were changed to DMEM-F12 (1:1) medium containing N2 supplements (Gibco), 20 ng/ml FGF2 (Millipore), 100 ng/ml FGF8 (Peprotech) and designated factors, including Shh (Peprotech), Wnt1 (Peprotech), Wnt5a (R&D), and GSK3β inhibitor CT99021 (Axon Medchem) before they were fixed with 4% PFA. The number of mature DA neurons in culture were determined by counting the total number of TH+ neurons per 20x field (Parish et al., 2008
Mouse Embryonic Stem Cell (mESC) Cultures
Differentiation of R1 mESCs into DA neurons was performed using a slightly modified protocol (Barberi et al., 2003
). Briefly, R1 mESC were seeded at a density of 50 cells/cm2
on mitomycin-treated stromal cells PA6 and cultured in ES-Serum Replacement Media, composed by KnockOut-DMEM (Gibco), 15% KnockOut serum replacement (Gibco), 0.1 mM β-mercaptoethanol (Sigma), 200 mM L-Glutamine (Gibco), 1% nonessential amino acids (Biochrom AG), 2,000 U/ml penicillin/streptomycin (Gibco). After 5 days, medium was changed and supplemented with 25 ng/ml FGF8 (R&D), and different concentrations of Shh (R&D) and GSK3β inhibitor CT99021. From day 8 to day 11, cells were cultured in N2 medium consisting of F12 and MEM mixture 1:1 (Gibco), Glucose, N2 supplement (Gibco), 15 mM HEPES (Gibco), 200 mM L-Glutamine, and 3 mg/ml AlbuMax I (Gibco) supplemented with 50 ng/ml FGF8 and 10 ng/ml FGF2 (R&D), and the same concentration of Shh and CT99021 as in days 5–8. From days 11–14, the media was replaced with N2 medium supplemented with 30 ng/ml brain derived neurotrophic factor (BDNF), 30 ng/ml glial derived neurotrophic factor (GDNF, both from R&D), and 200 µM ascorbic acid (Sigma).
After in vitro
differentiation, cells were fixed in 4% PFA (10’ RT), serum blocked and incubated in the appropriate primary and subsequently secondary antibodies as described previously (Parish et al., 2005
). Nuclear counterstaining was performed using Hoechst. The following antibodies were used: mouse monoclonal anti-III-tubulin (TuJ1; 1:500, Promega, Madison, WI), rabbit polyclonal anti-tyrosine hydroxylase (1:500, Pel-Freez, Rogers, AK), mouse monoclonal anti-tyrosine hydroxylase (1:500, ImmunoStar), rabbit anti-Foxa2 (1:500, Cell Signaling Technology), rabbit anti-Pitx3 (1:100, Zymed Laboratories, Inc.), rabbit anti-Nurr1 (1:500, Santa Cruz Biotechnology, Inc.), Alexa Fluor 488 goat anti-mouse and Alexa Fluor 555 donkey anti-rabbit (1:500, Molecular Probes, Eugene, OR).
Data were analyzed by two-tailed Student’s t test. Values were expressed as mean ± s.e.m. Changes were identified as significant if the p value was less than 0.05.