To generate Lgi4LacZ/LacZ
) mice, bacterial artificial chromosome (BAC) clones containing the Lgi4
genomic locus were purchased (Invitrogen) and a targeting vector was constructed using bacterial recombineering (Copeland et al., 2001
; Liu et al., 2003
). Bruce 4.G9 ES cells (a subline of Bruce4 selected for improved genetic stability (Kontgen et al., 1993
; Hughes et al., 2007
)) were electroporated with the targeting construct, positively selected with G418 (Gibco, Grand Island, NY), and negatively selected with gancyclovir (cytovene from Syntex; see Suppl. Fig. 1
for the targeting strategy). Correctly targeted ES cell clones were identified by Southern blot, and their chromosome numbers were confirmed to be euploid. Three independent ES cell clones were injected into blastcysts obtained from B6(Cg)-Tyrc-2J
/J mice (Jackson laboratory). The resulting male ES cell/mouse chimeras were crossed with B6(Cg)-Tyrc-2J
/J mice to obtain germline transmission. After germline transmission, the neo cassette was removed by crossing with B6.Cg-Tg(ACTFLPe)9205Dym/J mice (Rodriguez et al., 2000
mice and Adam22−/−
mice (Sagane et al., 2005
) were housed at the University of Michigan Unit for Laboratory Animal Medicine, an AAALAC accredited facility that follows the National Research Council’s guide for the care and use of laboratory animals.
Cell culture and self-renewal assay
Neural crest stem and progenitor cells were isolated and cultured as described in prior studies (Molofsky et al., 2005
; Joseph et al., 2008
; Nishino et al., 2008
). For adherent cultures, PNS cells were enzymatically dissociated and plated at clonal density (0.33 cells/µl = 500 cells per 35mm well), in 6 well plates (Corning) that had been sequentially coated with 150 µg/ml poly-d-lysine (Biomedical Technologies, Stoughton, MA) and 20 µg/ml laminin (Sigma). For the non-adherent culture of neurospheres, PNS cells were plated at 0.67–1.33 cells/µl (1000–2000 cells per 35mm well) in ultra-low binding 6-well plates (Corning). Cells were initially cultured for 8 to 10 days in ‘self-renewal medium’ to promote the formation of undifferentiated colonies. This medium contained a 5:3 mixture of DMEM-low:neurobasal medium, supplemented with 20 ng/ml recombinant human bFGF (R&D Systems, Minneapolis, MN), 20 ng/ml IGF1 (R&D Systems), 15% chick embryo extracts (CEE), 1% N2 supplement (Gibco), 2% B27 supplement (Gibco), 50 mM 2-mercaptoethanol, 35 µg/ml (110 nM) retinoic acid (Sigma), and penicillin/streptomycin (Biowhittaker). Cultures were subsequently refed with ‘differentiation medium’ and allowed to grow for an additional 4 to 7 days. Differentiation medium contained 10 ng/ml (instead of 20 ng/ml) bFGF, 5% fetal bovine serum (Gibco), no IGF, and no CEE. After being grown in self-renewal medium, neurospheres were routinely transferred to adherent cultures containing differentiation medium before being stained to assess multilineage differentiation (see below). All cultures were maintained at 37°C in 6% CO2
To measure self-renewal, individual PNS neurospheres were replated (one/well) for 72 h onto adherent plates to allow the spheres to spread out over the culture dish, facilitating dissociation. Individual adherent colonies were then dissociated with trypsin and collagenase (four parts 0.05% trypsin-EDTA plus one part 10 mg/mL collagenase IV) for 3 min at 37°C followed by trituration. Two thousand dissociated cells were replated per well of a six-well plate. Secondary neurospheres were cultured, differentiated, and the number of multilineage secondary colonies generated by each primary colony were counted.
Isolation of neural tissues
E13.5 embryonic PNS tissues (DRG, sympathetic chain, and gut) were dissected into ice cold PBS and dissociated by incubating for 4 min at 37°C in trypsin/EDTA (BioWhittaker, product 17-161E, diluted 1:10 in Ca, Mg-free HBSS) plus 0.25 mg/ml type 4 collagenase (Worthington, Lakewood NJ). P0 gut plexus/outer muscle layer cells were minced, and dissociated for 15 min at 37°C in 0.025% trypsin/EDTA (Gibco 25300-054, Grand Island, NY) plus 1 mg/ml type 4 collagenase (Worthington) in Ca, Mg-free HBSS. After centrifuging, the cells were gently triturated, filtered through nylon screen (45 mm, Sefar America, Kansas City, MO), counted by hemocytometer, and resuspended in staining medium before being sorted or plated. Staining medium was L15 medium containing 1 mg/ml BSA (Sigma A-3912, St. Louis, MO), 10 mM HEPES (pH 7.4), and penicillin/streptomycin (BioWhittaker, Walkersville, MD).
Antibodies and immunohistochemistry
PNS tissues were fixed in 4% paraformaldehyde overnight, then cryoprotected in 30% sucrose, embedded in OCT compound and frozen. 10 µm sections were cut, then pre-blocked for 1 hr at room temperature in blocking solution (PBS containing 5% goat serum, 0.2% bovine serum albumin, and 0.5% TritonX-100), incubated with primary antibody at 4°C overnight, followed by washing, and incubation in secondary antibody for 1 hr at room temperature. Primary antibodies included those against Krox20 (Covance, Berkeley, CA, PRB-236P, 1:1000), Periaxin (gift from Dr. P. Brophy, University of Edinburgh, UK 1:400) (Gillespie et al., 1994
), Peripherin (Millopore, Billerica, MA, 1:1000), beta-galactosidase (gift from Dr. T. Glaser, University of Michigan), Tuj1 (Covance, Berkeley, CA, MMS-435P, 1:1000), BFABP (gift from Dr. T. Muller, Max-Delbruck-Center, Berlin, Germany, 1:2000) (Kurtz et al., 1994
), p75 (Millipore, AB1554, 1:1000), Sox10 (R&D Systems, 20B7, 1:400), GFAP (BD Pharmingen, San Diego, CA, 1:400), phospho-Histone H3 (Cell Signaling Technology Inc., Danvers, MA, 1:200), HuC/D (1:200, Molecular Probes, Inc., Eugene, OR), and NeuN (Millipore, MAB377,1:1000). For secondary antibodies, Alexa-Flour 488 or 555 conjugated antibodies were used (Molecular Probes, Inc., Eugene, OR, 1:1000). Slides were counter stained in 2.5µg/ml DAPI for 10 min at room temperature, then mounted using ProLong antifade solution (Molecular Probes, Eugene, OR).
For in situ hybridization to detect Adam9, Adam11, Adam22, and Adam23 transcripts, E14.5 embryos or P0 pups were fixed in 4% paraformaldehyde overnight, cryoprotected in 30% sucrose, embedded in OCT compound, and frozen. 10 µm sections were cut, pretreated with 2µg/ml Proteinase K at 37°C for 20 min, with 0.2N HCl for 10 min at room temperature with 0.1M triethanolamine-HCl for 10 min at room temperature and hybridized with Digoxigenin-labeled Adam antisense probe at 55°C overnight. The next day, sections were washed with 2XSSC for 30 min at 55°C, with 0.2XSSC for 40 min, blocked with 20% goat serum for 1 hr, and incubated with anti-Digoxigenin-AP (Alkarine-Phosphatase) Fab fragment (1:2000, Roche) for 60 min at room temperature. Sections were washed with Tris buffered saline (pH 9.5) with 0.1% tween-20 for 30 min at room temperature, and incubated with 0.5µl/ml NBT (nitro-blue tetrazolium chloride) plus 3.5µl/ml BCIP (5-Bromo-4-Chloro-3'-Indolylphosphatase p-Toluidine salt) (Roche).
For X-gal staining, E10.5 and E13.5 mouse embryo or P0 pups were fixed with 1% paraformaldehyde/0.2% glutaraldehyde for 10 min at 4°C. Then whole embryos (E10.5) or cryosections (E13.5 and P0) were incubated in staining solution at 37°C for 4–16 hr.
Genotyping was performed by PCR using GoTag Flexi DNA polymerase (Promega), following the manufacturer’s instructions. The primers for Lgi4 genotyping were: 5’-GCATCCCACGGAGATGTAGT-3’ (common sense primer), 5’-CAACCTGCACCTTTCCAAAT-3’ (antisense primer for the detection of the wild-type allele), and 5’-GTTGTGGCGGATCTTGAAGT-3’ (antisense primer for the detection of the Lgi4LacZ allele). Primers for Adam22 genotyping: 5’-TGAGTTGGGCAGAACTGAGTCACTG-3’ (common sense primer), 5’-AGGAATTGCAAAGAAGAGCCTGTGAC-3’ (antisense primer for the detection of the wild-type allele), and 5’-CATGCTCCAGACTGCCTTGGGAAAAG-3’ (antisense primer for the detection of Adam22− allele). The PCR cycle was 94°C for 2 min, followed by 35 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1min, and followed by 72°C for 5 min.
PNS neurospheres were replated into adherent cultures in which the plates had been coated with poly-D-lysine and laminin. The neurospheres were allowed to differentiate for 7 to 9 days, then fixed in acid ethanol for 20 min at −20°C, washed, blocked, and triply labeled with antibodies against peripherin (Chemicon International, AB1530, 1:1000), GFAP (Sigma, G-3893, 1:200), and SMA (Sigma, A-2547, 1:200) as described previously (Kruger et al., 2002
). Cells were stained for 10 min at room temperature with 10 µg/ml DAPI (Sigma, D-8417) to visualize nuclei.
To study the proliferation of glial progenitors, cells were fixed in 70% ethanol for 30 minutes at −20°C, and stained with antibody against phospho-Histone H3 (Cell Signaling Technology, 9701, 1:100). Cells were counterstained for 10 min at room temperature with 10 µg/ml DAPI (Sigma D-8417).
For X-gal staining of neurospheres, E13.5 PNS neurospheres were fixed with 1% paraformaldehyde/0.2% glutaraldehyde for 5 min at 4°C and incubated for 1 hr at 37°C in staining solution: PBS containing 2 mM 5-bromo-4 chloro-3-indolyl-beta-D-balactoside (X-gal; Molecular Probes, Eugene OR, USA), 2 mM MgCl2, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, and 0.02% NP-40. In some cases, neurospheres were transferred to plates coated with poly-D-lysine and laminin, and cultured for 3 days in self-renewal medium. Cells were fixed with 1% paraformaldehyde for 5 min at 4°C, incubated in staining solution at 37°C for 1 hr to assess LacZ expression. Cells were then blocked, stained with anti-Nestin antibody (Millipore, MAB353, 1:400) for 1hr at room temperature, followed by Alexa-Flour 488 conjugated anti mouse IgG1 antibody (Molecular Probes, Inc., Eugene, OR, 1:500).
Nerves were dissected and fixed in 2.5% glutaraldehyde overnight at 4°C. Nerves were rinsed with PBS and re-fixed in 1% OsO4
. (Electron Microscopy Sciences, Fort Washington PA, USA) for 1hr at room temperature. Nerves were then dehydrated in an ethanol series (30%, 50%, 70%, 95%, and 100% ethanol) before infiltrating and embedding in Spurr resin(Bozzola and Russell, 1992
). Semithin sections (1 µm) were cut with a glass knife. Thin sections (70 nm) were cut with a diamond knife. Some sections were stained with uranyl acetete to reveal the basal lamina. Sections were examined with a transmission electron microscope (Phillips CM-100).
For immunoprecipitation, 293T cells were co-transfected with Lgi-4-FLAG and HA-tagged ADAMs (ADAM9-HA, ADAM11-HA, ADAM22-HA, and ADAM23-HA; gift from Dr. M. Fukata, NINS, Japan). 24hr after transfection, cells were harvested and suspended in lysis buffer (20mM Tris, 137mM NaCl, 10% glycerol, 1% NP-40, 2mM EDTA, and protease inhibitor cocktail (complete mini-tablet, Roche)) for 1 hr at 4°C, incubated with ProteinG-Agarose at 4°C for 60 min to absorb non-specific binding. After eliminating ProteinG-Agarose by centrifugation, small fractions of supernatants were saved as input, and the rest was incubated overnight at 4°C with either anti-FLAG antibody (M2, Sigma, 1:500) or anti-HA antibody (Sigma, H6908, 1:500). The next day, immunoprecipitated fractions were harvested by incubating with ProteinG-Agarose for 60 min at 4°C, washed with washing buffer, and boiled for 5 min with SDS sample loading buffer. SDS PAGE was done in 4–20% Tris-Glycine Gels (Invitrogen) and transferred to PVDF membranes (Millipore). The membranes were blocked in Tris buffered saline with 0.05% Tween-20 and 5% milk, incubated with primary and secondary antibodies, and washed following standard procedures. Horseradish peroxidase conjugated secondary antibodies were detected by chemiluminescence (ECL Plus; Amersham-Pharmacia).
For the cell surface binding assay, COS7 cells were seeded onto poly-d-lysine coated coverslips, and co-transfected with Lgi-4-FLAG and HA-tagged ADAMs. 24hr after transfection, cells were fixed with 2% paraformaldehyde for 10 min at 4°C, and blocked with PBS containing 2mg/ml BSA for 10min at 4°C. Cells were immediately stained with anti-FLAG antibody (1:1000), followed by Alexa-Flour 488 conjugated anti-mouse IgG1 antibody (Molecular Probes, Inc., Eugene, OR, 1:500). Then cells were permeabilized with 0.1% Triton X-100 for 10 min, blocked, and stained with anti-HA antibody (1:1000), followed by Alexa-Flour 555 conjugated anti mouse IgG1 antibody (Molecular Probes, Inc., Eugene, OR, 1:500).