BALB/cJ and C57BL/6J inbred mouse strains were purchased from Jackson Laboratories (Bar Harbor, ME). Engineered mice with a LacZ cassette replacing the endogenous Sox10 locus (Sox10LacZ/+; Britsch et al., 2001) were obtained on a mixed genetic background and maintained at NHGRI by crossing to C57BL/6J for several generations. All other mice described in the ENU screen were bred and housed in the NHGRI animal facility according to NIH guidelines. For genotyping, genomic DNA was prepared from tail biopsies or yolk sacs using a PUREGENE DNA purification kit (Gentra Systems, Inc. Minneapolis, MN) according to the manufacturers instructions. Noon on the day of vaginal plug observation was designated E0.5 for timed pregnancies. Reabsorbed embryos were counted as dead in analysis of lethality data.
ENU Treatment and Mutagenesis Screen
ENU was prepared and injections were carried out as previously described 
.Briefly, ENU (Sigma; St. Louis, MO) was dissolved at 100 mg/ml in 95% ethanol and diluted to 5 mg/ml in a sterile phosphate/citrate buffer (0.1 M dibasic sodium phosphate, 0.05 M sodium citrate, pH 5.0). A spectrophotometer reading at a wavelength of 398 nm was used to confirm the ENU concentration, and BALB/cJ male mice were given weekly intraperitoneal injections at 0.1 mg per gram of body weight for three consecutive weeks. Mice were allowed to recover for eight weeks post-injection and loss of fertility was confirmed by mating to C57BL/6J females. Males that lost and subsequently regained fertility (G0
) were bred to C57BL/6J females beginning at 12 weeks post-injection and the resulting first generation male progeny (G1
) were crossed to Sox10LacZ/+
females. In the next generation, female Sox10LacZ/+
) were backcrossed to their G1
fathers, and embryos (G3
) were harvested at E11.5 for β-galactosidase staining.
The number of samples and markers used in the initial genome scan for each pedigree is provided in . In each case DNA from affected G3 embryos and G2 obligate carriers was used for genotyping at simple sequence length polymorphism (SSLP) or single nucleotide polymorphism (SNP) markers spaced throughout the genome. The markers were polymorphic between the BALB/cJ and C57BL/6J parental strains used in this study and were genotyped by gel electrophoresis following PCR amplification (SSLPs) or using the Sentrix Universal Array Matrix platform (Illumina, San Diego, CA) (SNPs). Following the initial genome scan, additional markers from regions of significant linkage were used to refine the location of each msp locus and genotype additional G2 and G3 animals. Subsequently, markers flanking each locus () were used to identify viable heterozygous carriers and maintain the line during six or more subsequent generations of outcrossing to C57BL/6J.
Using a candidate gene approach, Erbb3 was chosen for sequencing in the msp1 pedigree. Each exon and surrounding splice sites were amplified from genomic DNA for sequencing using standard techniques (Harvard Partners Healthcare Center for Genetics and Genomics, Harvard Medical School). The sequence of all Erbb3 coding exons from an affected homozygote msp1 mutant was compared to sequence from the parental inbred strains to identify a single nucleotide change on the ENU treated chromosome.
TaqMan Assay for msp1 Genotyping
Taqman MGB probes (Applied Biosystems, Foster City, CA) were designed across the site of the msp1 mutation specific for both the wild-type allele (FAM-TGG-AAG-TAG-a-TAA-GAA-TG) and the ENU induced mutant allele (VIC-TGG-AAG-TAG-g-TAA-GAA-T). PCR reactions were carried out in 2× GenAmp PCR Master Mix, 10 µM each primer (primer 1: AGG-GCT-TGT-CCT-GCT-GAC-AA and primer 2: TGC-AAG-GCT-CAC-ACA-TCT-TGA), and 100 nM each allele-specific probe. Cycling conditions were 2 minutes at 50°C, 10 minutes at 95°C, followed by 40 cycles of 92°C for 15 seconds and 60°C for 1 minute. Relative quantitation of the two alleles was determined in an end-point assay for genotyping.
Dissected embryos were fixed in 1× phosphate buffered saline (PBS), 1% formaldehyde, 0.2% glutaraldehyde, 0.02% NP-40 for 2 hours on ice, and X-gal staining was performed as previously described 
Whole-Mount In Situ Hybridization
Whole-mount in situ
hybridization was performed using digoxigenin-labeled antisense probes as previously described 
. Reverse-transcribed digoxigenin-conjugated probes were made from linearized plasmids with polymerase binding site linkers (all reagents from Roche Diagnostic Incorporation, Indianapolis, IN). The following DNA sources were used for probe synthesis: 1.5-kb Sox10
(nucleotides 1287–2787, dcgs10 BssHII
T7 RNA polymerase), and Erbb3 
, T7 RNA polymerase).
Embryonic Cell Culture
Timed pregnancies between Erbb3msp1/+ heterozygote mice were established and embryos were dissected out at E11.5. The yolk sac was stored for DNA isolation and used for genotyping of the msp1 mutation. Dissected embryos were dissociated using a sterile razor blade, and digested in 0.05% Trypsin-EDTA for 30 minutes at 37°C. The trypsin digestion was inactivated with DMEM medium supplemented with 10% fetal bovine serum. To obtain single-cell suspension, digested embryos were pipeted up and down several times using a 2-ml pipet. The cell suspensions were transferred to a 15 ml conical tubes and spun down for 5 minutes at 1200×g. Supernatant was carefully removed, and cell pellet was resuspended in fresh DMEM medium supplemented with 10% fetal bovine serum, 10 nM Endothelin 3 (Sigma), 50 ng/ml Stem cell factor (R&D Systems, Minneapolis, MN) and 10 ng/ml Wnt-3a (R&D Systems) and cultured for 24 hours at 37°C and 5% CO2. The following day, cultures were stimulated with 5 ng/ml NRG1-β1 (R&D Systems) for 15 minutes and lysed in RIPA lysis buffer [1XTBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.004% sodium azide] supplemented with 2 mM sodium orthovanadate, 4 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail (Santa Cruz Biotechnology).
Molecular modeling of the mouse ERBB3 extracellur region was performed using the MODELLER package 
as implemented within Discovery Studio (Accelrys, San Diego, CA). Modeling was based on the solved structure of human ERBB3, as determined at a resolution of 2.6 Å by X-ray diffraction (pdb|1M6B) 
. The mouse sequence was aligned with the human template sequence using Clustal W. The two sequences share 92% residue identity over the region modeled. To generate wild type and mutant structures, the MODELLER program was run in a fully-automated mode with a high optimization level in order to construct energy-minimized three-dimensional models of the aligned target sequences by satisfaction of spatial restraints extracted from the template PDB file.
Whole embryo lysates were prepared as follows. E11.5 embryos were dissected out and homogenized in RIPA lysis buffer supplemented with 1 mM Na4
, 2 mM PMSF and protease inhibitor cocktail (Santa Cruz Biotechnology, Santa Cruz, CA) followed by a brief sonication. The lysates were cleared by centrifuged at 14,000×g for 10 minutes at 4°C, and protein concentration of the supernatant was determined using a BCA protein assay kit (Pierce, Rockford, IL). Cell lysates containing equal amounts of protein were separated on 8% Tris-Glycine SDS gel (Invitrogen, Carlsbad, CA), transferred to polyvinylidene fluoride membrane (Invitrogen) and blocked with 5% milk in TBST [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 20] for 1 hour at room temperature. The membranes were then incubated with primary antibody against ERBB3 (1
1000 diluted in 5% milk in TBST) (Santa Cruz Biotechnology; C-17), phospho-ERBB3 (1
500 diluted in 5% bovine serum albumin (BSA) in TBST) (Cell Signaling, Santa Cruz, CA; 21D3), α-Tubulin (1
1000 diluted in 5% milk in TBST) (Sigma) at 4°C over night. After washing in TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1
10,000) (Amersham Biosciences, Little Chalfont, UK) for 1 hour at room temperature. The protein bands were detected using an enhanced chemiluminescence kit (Millipore, Billerica, MA).
Cell Surface Biotinylation Assay
Embryonic cell cultures were prepared as described above. On day two, cultures were stimulated with NRG1-1β for 10 minutes, washed twice in 1× PBS and chilled on ice for 5–10 minutes. Cell surface proteins were labeled with 1 mg/ml EZ-Link Sulfo-NHS-SS-Biotin (Pierce, Rockford, IL) in PBS for 30 minutes at 4°C with gentle shaking. Biotin was aspirated and cultures were washed twice for 3–5 minutes with 100 mM Tris, pH 8.0 in PBS followed by one wash in 1× PBS. Cells were solubilized in mild-lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 1% NP-40, 10% glycerol) supplemented with 1 mM Na4P2O7, 2 mM PMSF and protease inhibitor cocktail (Santa Cruz Biotechnology, Santa Cruz, CA) for 30 minutes at 4°C on a shaker, and centrifuged for 15 minutes at 10,000×g. Supernatants were removed and the protein concentration determined by a BCA protein assay kit (Pierce, Rockford, IL). An equal amount of protein (1 mg) of each genotype was precipitated with 100 ul NeutrAvidin agarose beads (Pierce, Rockford, IL) overnight at 4°C with rotation. Beads were washed three times in mild-lysis buffer, and surface proteins were eluted in 50 ul 2× SDS loading buffer supplemented with 50 mM DTT (Invitrogen, Carlsbad, CA). Equal volumes (40 ul) of each genotype were separated on 8% Tris-Glycine SDS gel, and membranes were blotted with anti-ERBB3 and/or phospho-ERBB3 antibody.
The msp1 mutation was introduced in the human Erbb3 cDNA (Clone ID #6147464, Open Biosystems, Huntsville, AL) via QuickChange II Site-Directed Mutagenesis Kit according to manufacturer's instructions (Stratagene, La Jolla, CA). The following PCR primers were used to introduce the mutation: Fwd-CCTGACAAGATGGAAgTAGGTAAAAATGGGCTCAAGATG- and Rev-CATCTTGAGCCCATTTTTACCTACTTCCATCTTGTCAGG-. The presence of the msp1 mutation was confirmed by sequencing.
Cell Culture and Transfection
293T cells were cultured in DMEM medium with 10% fetal bovine serum, 100 units/ml penicillin and 100 ug/ml streptomycin at 37°C in a humidified 5% CO2. Erbb3msp1 and Erbb2 (Clone ID #6178526, Open Biosystems) or wild-type Erbb3 and Erbb2 clones were co-expressed in 293T cells using FuGENE 6 (Roche Applied Science) transfection reagent. 48 hours post-transfection, cells were starved overnight in serum-free DMEM. The following day, cells were stimulated with or without 5 ng/ml NRG1- β1 (R&D Systems) for 15 minutes. Cell lysates were collected in RIPA lysis buffer and analyzed by Western blotting.