Breeding and genotyping mice
mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Conditional Met mutant mice (Emx1cre
) were generated by mating mice homozygous for a Met allele, in which exon 16 is flanked by loxP sites (Huh et al., 2004
, courtesy of Dr. Snorri Thorgeirsson, NIH/Center for Cancer Research, Bethesda, MD) to Emx1-cre mice (Gorski et al., 2002
) (courtesy of Dr. Kevin Jones, University of Colorado, Boulder, CO) that were also heterozygous for the floxed allele (Emx1cre
). Both Metfx/fx
breeding lines were back-crossed onto the C57BL/6J
background for greater than 10 generations, and their progeny (i.e., Emx1cre
and littermate control mice), were genotyped via polymerase chain reaction (PCR). The PCR primer sets were as follows: Emx1cre
forward 5′-TTCGGCTATACGTAACAGGG-3′ and reverse 5′-TGCATGCAACGAGTGATGAG-3′; Metfx
forward 5′-GCAACTGTCTTTTGATCCCTGC-3′ and reverse 5′-TGTCCAGCAAAGTCCCATGATAG-3′. For the Emx1cre
reaction, DNA samples are submitted to an initial denaturation step of 5 minutes at 94°C, then 35 amplification cycles [(denaturation: 94°C for 45 seconds), (annealing: 55°C for 30 seconds), (elongation: 72°C for 1 minute)], and then a final elongation step of 5 minutes at 72°C. The expected PCR product size is 350 bp. For the Metfx
reaction, DNA samples are submitted to an initial denaturation step of 5 minutes at 94°C, then 35 amplification cycles [(denaturation: 94°C for 45 seconds), (annealing: 60°C for 1 minute), (elongation: 72°C for 2 minutes)], and then a final elongation step of 5 minutes at 72°C. The expected PCR product size is 500 bp for the wild type Met
allele and 580 bp for the Metfx
A cross-sectional approach was employed to assess patterns of Met transcript and total Met protein expression in the developing mouse forebrain. For each experimental methodology described, forebrains from at least 3 mice from at least two independent litters were analyzed at each developmental time point of interest. In the case of immunohistochemical studies requiring comparisons of Emx1cre/Metfx/fx and littermates, at least 3 experimental pairs from independent litters were analyzed per time point.
All research procedures using mice were approved by the Institutional Animal Care and Use Committee at Vanderbilt University and conformed to NIH guide-lines. All efforts were made to minimize animal suffering and to reduce the number of animals used.
In situ hybridization
Two cDNA probe templates specific to the mouse Met gene were generated by RT-PCR: a 1,387 bp fragment corresponding to nucleotides 2665-4051 of NM_008591 and a 1,404 bp fragment corresponding to nucleotides 37-1440 of NM_008591. Antisense and sense cRNA probes were transcribed from these templates with incorporation of 35S-CTP.
Pregnant dams were deeply anesthetized with isofluorane, fetuses were harvested and the dissected brain rapidly removed, frozen in isopentane, and stored at -80° C until cryosectioning at 20 μm. Cryosections were thaw-mounted onto Superfrost Plus glass slides (Fisher) and stored at -80°C.
Slide-mounted sections were postfixed in buffered 4% paraformaldehyde for 15 minutes, washed in 0.1 M phosphate-buffered saline (PBS), pH 7.4, for 5 minutes, acetylated with 0.25% acetic anhydride in 0.1M triethanolamine-HCl/0.15 M NaCl with 0.16%HCl for 10 minutes, and washed in 2X standard saline citrate (SSC; 0.15 M NaCl, 0.015 M sodium citrate) for 1 minute. Tissue was then dehydrated in graded alcohols (50%, 70%, 95%, 100%; 1 minute incubation each), delipidated in chloroform (two 5 minute incubations), and incubated in 100% and 95% ethanol (1 minute each). Slides were dried at 37°C for at least 2 hours.
Each slide was hybridized with 3 ng radiolabeled probe in 100 μl hybridization buffer (50% formamide, 0.75 M NaCl, 20 mM 1,4-piperazine diethane sulfonic acid, 10 mM EDTA, 10% dextran sulfate, 5X Denhardt’s solution, 50 mM DTT, 0.2% sodium dodecyl sulfate, 100 μg/ml sonicated salmon sperm DNA, and 10 μg/ml yeast tRNA) per slide. Hybridization was performed at 55°C for 16 hours in a humid chamber.
Following hybridization, coverslips were removed in 4X SSC plus 390mM 2-mercaptoethanol and slides were incubated in this solution for 15 minutes at room temperature followed by 4X SSC without 2-mercaptoethanol for 15 minutes at room temperature. Sections were then treated with 1:1 formamide/buffer (0.6M NaCl, 40 mM Tris base, 2 mM EDTA, 0.06% HCl) at 60°C for 30 minutes and washed in room temperature 2X SSC for 5 minutes. Sections were then treated with 20ug/ml RNase A in 0.5 M NaCl, 10 mM Tris, pH 8.0, and 1 mM EDTA at 37°C for 30 minutes. The slides were then washed in graded salt solutions (2X, 1X, and 0.5X SSC each for 5 minutes at room temperature, and 0.1X SSC at 65°C for 30 minutes). Slides were cooled to room temperature in 0.1X SSC for 5 minutes and dipped in 60% ethanol with 0.33 M ammonium acetate. Slides were dried at 37°C for 3-6 hours and exposed to X-ray film (Biomax MR; Eastman Kodak, Rochester, NY) for 3-15 days. A subset of sections were prepared for emulsion dipping, following the protocol noted in (Campbell and Levitt, 2003
) and exposed for 3-6 days prior to development.
The primary antibody used for Met immunohistochemical study was mouse anti-Met (Met, B-2; sc-8057; lot No. C2807; Santa Cruz Biotechnology, Santa Cruz, CA; immunogen: peptide corresponding to amino acids 1330-1379 of mouse Met (NCBI# NP 032617)). Using immunoblotting methods, the antibody recognizes the recombinant Met protein (Santa Cruz), and a minor band at 170 kD and a major band at 140 kD in brain tissue homogenates (see below). These bands represent pre-processed and processed forms, respectively, of the Met receptor. Only the pre-processed band was detected in homogenates prepared from mouse neocortex in which the Met
gene was deleted from the dorsal pallium (data not shown). A mouse monoclonal antibody, 1G9, generated in the laboratory against adult mouse hippocampal homogenates, cross-reacts specifically with phosphorylated neurofilament-H (NF-H) (Pennypacker et al., 1991
), and was used to stain developing axons.
Postnatal mice were deeply anesthetized with sodium pentobarbital (60 mg/kg i.p.) prior to transcardial perfusion with room temperature phosphate-buffered 4% paraformaldehyde (pH 7.3) containing 1.3% L-lysine and 0.24% sodium periodate. After postfixation overnight at 4°C, brains were cryoprotected via sequential 12-hour incubations in 10%, 20%, and 30% sucrose in PBS, pH 7.5. Fetal brains were harvested and immersion-fixed overnight at 4°C prior to cryoprotection.
Fetal and P0 fixed brains were frozen in embedding medium (Triangle Biomedical Sciences; Durham, NC) over liquid nitrogen vapors and stored at -80°C until sectioned with a cryostat at 20 μM. Sections were collected on gelatin-subbed slides and stored at -80°C until processed. Fixed brains from P7 through P35 were frozen, cut at 40 μM with a sliding microtome (Leica, Bannockburn, IL) and free-floating sections were stored in a cryopreservative solution at -20°C until processed. One series of sections from selected brains were stained with Cresyl Violet as previously described (Hockfield, 1993
For Met immunohistochemical processing, both cryostat and free-floating sections were rinsed in PBS and then incubated for 5 minutes in 0.5% H202 in PBS to quench endogenous peroxidases. The sections were then rinsed in PBS before 25 min incubation in 0.1 M Tris-glycine (pH 7.4). Several more PBS rinses preceded a 1.5 hr incubation in unlabeled donkey anti-mouse IgG (Fab; Jackson Immunoresearch, West Grove, PA) to block endogenous mouse immunoglobulins. Sections were further blocked in several rinses of Blotto-T (4% Carnation dried milk in PBS containing 0.2% Triton-X-100). Blocked sections were incubated in primary mouse anti-Met antibody diluted 1:250 in Blotto-T. Cryosections were incubated for 2-4 hours at room temperature; free-floating sections were incubated for 48-72 hours at 4°C. Following washes in Blotto-T, sections were incubated for 1 hour at room temperature in 1:1000 biotin-SP-conjugated donkey anti-mouse IgG (Jackson Immunoresearch) diluted in Blotto-T. Sections then were rinsed several times in PBS and processed by the ABC Elite histochemical method (Vector, Burlingame, CA). Met-specific antibody complexes were visualized by incubating the sections for 2-4 minutes at room temperature in 0.5% 3′3′-diaminobenzidine (DAB) with 0.015% H202. The stained sections were rinsed in PBS, and free-floating sections were mounted on gelatin coated slides. Finally, sections were dehydrated with ethanol, cleared with xylene, and coverslipped in DPX (Fisher, Pittsburgh, PA) for microscopic analysis. Phosphorylated NF-H staining was performed using a similar immunohistochemical protocol for free-floating sections, but with the following specific parameters: 1) The 1G9 primary antibody was diluted 1:200 in Blotto-T, 2) biotin —SP-conjugated donkey anti-mouse IgM secondary antibodies were diluted 1:1,000 in Blotto-T, and 3) antigen/antibody complexes were visualized using standard ABC reagents (Vector), followed by DAB histochemistry.
The following primary antibodies were used for Western blotting studies: rabbit anti-Met (Met; # 07-283; lot No. 27208; Millipore (Upstate), Billerica, MA; immunogen: peptide corresponding to amino acids 1361-1379 of mouse Met (NCBI# NP 032617)), mouse anti-Met (Santa Cruz, sc-8057), mouse anti-alpha-tubulin loading control (alpha-tubulin, Ab-1; # CP06; lot No. D16509-5; Oncogene Research Products, San Diego, CA; immunogen: native chick brain microtubules), mouse anti-GAPDH loading control (GAPDH; #AM4300; lot No. 08608176A; Ambion, Austin, TX; immunogen: purified rabbit muscle GAPDH).
Mice were deeply anesthetized with sodium pentobarbital (60 mg/kg i.p.) prior to decapitation and brain removal. Harvested brains were immediately immersed in room temperature Hanks’ balanced salt solution (Sigma, Saint Louis, MO). With the aid of an MZ-6 stereozoom microscope (Leica), the cerebral cortex from each hemisphere was divided evenly midway along the antero-posterior axis to generate two tissue samples. The underlying striatum also was rapidly dissected before all samples were snap-frozen on liquid nitrogen and stored at -80°C.
P0 through >P90 cortical and striatal samples were prepared by homogenizing frozen tissue samples in a glass tissue homogenizer (Wheaton, Millville, NJ) with ice-cold homogenization buffer (50 mM Tris HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% deoxycholate, 0.5mM DTT , 2 mM EDTA, pH 8.0, 2 mM EGTA, 0.2mM PMSF) containing a protease inhibitor cocktail (Sigma), 50 mM activated Na3VO4, 100 nM microcystin, and 0.5 nM cypermethrin. E16 tissue was sonicated briefly in the same buffer. Tissue homogenates were cleared by a 16,000 × g centrifugation for 20 minutes at 4°C, and protein concentrations of the supernatants were determined using the Dc protein assay (Bio-Rad, Hercules, CA).
Protein samples (35 μg protein per lane) were fractionated by SDS-PAGE and transferred to supported nitrocellulose membranes. The membranes were then blocked for 1 hr at room temperature in Blotto (3% Carnation dried milk in PBS) before being incubated with primary antibodies. Polyclonal rabbit anti-Met antibodies and alpha-tubulin antibodies were diluted 1:2500 and 1:100,000, respectively, in Blotto containing 0.05% Tween-20. Monoclonal mouse anti-Met antibodies were diluted 1:500 in Blotto alone, and GAPDH antibodies were diluted 1:200,000 in Blotto + 0.02% Tween-20. The membranes were then rinsed repeatedly in PBS and incubated for 1.5 hr at room temperature with anti-rabbit and anti-mouse horseradish peroxidase-conjugated secondary antibodies (Jackson Immunoresearch). Following several more rinses in PBS, the membranes were reacted with enhanced chemiluminescence reagents (GE/Amersham ECL) and detected with autoradiography film (GE/Amersham hyperfilm). Autoradiographic films were imaged with a high resolution scanner and subjected to densitometric quantification using IMAGE-J.
Microscopy was performed with the aid of an Axioplan II microscope (Zeiss, Jena, Germany), and micrographs were acquired with a Zeiss AxioCam HRc camera (Zeiss) in Axiovision 4.1 software (Zeiss). No image alteration other than re-sizing was performed. Figures were prepared digitally in Microsoft Office Powerpoint 2003 (Microsoft Incorporated, Redmond, WA).