The following mouse lines were purchased from The Jackson Laboratory (Bar Harbor, ME): Cxcr4 (stock no. 004341), Z/EG cre reporter line (stock no. 004178), GAD1-green fluorescent protein (GFP) transgenic line (stock no. 003718), and ubiquitous inducible cre line CAG-CreER (stock no. 004682). Lhx6GFP BAC line was obtained from GENSAT (The Gene Expression Nervous System Atlas Project at The Rockefeller University, New York, NY). Cxcr4-flox animals were kindly provided by Dr. Dan Littman (New York University, New York, NY) and the Dlx5/6Cre line was generated in the laboratory of J. L. R. Rubenstein [University of California, San Francisco (UCSF)]. The following breeding schemes were used to obtain littermate controls and mutants: Lhx6-GFPTg/+;Cxcr4+/− × Cxcr4+/− (for Lhx6-GFPTg/+;Cxcr4+/+ and Lhx6-GFPTg/+;Cxcr4−/−), CAG-CreERTg/+;Cxcr4+/− × Cxcr4flox/flox (for CAG-CreERTg/+;Cxcr4flox/+ and CAG-CreERTg/+;Cxcr4flox/−), Z/EGTg/+;Dlx5/6CreTg/+;Cxcr4+/− × Cxcr4flox/flox (for Z/EGTg/+;Dlx5/6CreTg/+;Cxcr4flox/+ and Z/EGTg/+;Dlx5/6CreTg/+;Cxcr4flox/−), Lhx6-GFPTg/+;Dlx5/6CreTg/+;Cxcr4+/− × Cxcr4flox/flox (for Lhx6-GFPTg/+;Dlx5/6CreTg/+;Cxcr4flox/+ and Lhx6-GFPTg/+;Dlx5/6CreTg/+;Cxcr4flox/−), and GAD1-GFPTg/+;Dlx5/6CreTg/+;Cxcr4+/− × Cxcr4flox/flox (for GAD1-GFPTg/+;Dlx5/6CreTg/+;Cxcr4flox/+ and GAD1-GFPTg/+;Dlx5/6CreTg/+;Cxcr4flox/−). The day of vaginal plug was considered embryonic day 0.5 (E0.5). Mouse colonies were kept at UCSF in accordance with National Institutes of Health and UCSF guidelines. The Cxcr4-null mice are on a pure C57BL/6 background, but all other lines of mice are on mixed backgrounds between C57BL/6 and CD1. In all cases, controls are littermate and from the same degree of mixed background.
Cortical slice culture and bead analysis
We dissected brains from E13.5 embryos or postnatal day 0 (P0) newborns in ice-cold 1× Hanks buffer (no. 140-25-076; Invitrogen, Carlsbad, CA). Lhx6-GFP+ brains were selected by direct inspection with a fluorescence dissection microscope. The 250 μm coronal cortical sections were prepared by cutting on a Leica (Nussloch, Germany) vibrating microtome. Slices were grown on Millicell-CM (Biopore PICMORG50) in 35 mm Petri dishes in serum-free medium (1× Neurobasal medium, 2% B-27 supplement, 0.5% glucose, 1% penicillin/streptomycin, 2 mm GlutaMAX-1). Slices were allowed to recover for 2–3 h before any treatment was started. For bead analysis, we soaked agarose beads (Bio-Rad, Hercules, CA) with BSA (10 μg/ml; Sigma, St. Louis, MO) or Cxcl12 (SDF-1α) (10 μg/ml; R&D Systems, Minneapolis, MN) at 4°C for 1 h before placing the beads at the appropriate locations in the slices with fine forceps. After culture at 37°C with 5% CO2 for the listed amount of time, confocal pictures were taken for analyzing the chemoattractant effect.
In utero electroporation
Timed pregnant mice at desired ages were anesthetized with sodium pentobarbital at 60 mg/kg body weight. The abdomen was cleaned with 70% ethanol. A midline incision was made, and the uterus was exposed. The cerebral vesicle of each embryo was transilluminated with a fiber optic source and DNA solution prepared at 2 mg/ml in 10 mm Tris-HCl, pH 8.0, with 0.04% trypan blue was injected into lateral ventricle with a glass micropipette. After injection, embryos were held with a forcep-type electrode with two platinum paddles (Protech International, San Antonio, TX). Electrical pulses (42 V for E13.5, 50 V for E14.5) with 50 ms duration were delivered five times at 950 ms intervals using a square-pulse electroporator BTX830. After the procedure, the uterus was replaced into the abdominal cavity, and the abdominal cavity was filled with prewarmed 1× PBS. The abdominal wall and skin were sutured to allow the embryos to develop further. The mother was placed on a 37°C hot plate until recovery from the surgery. The entire surgical procedure was completed within 45 min. The following full-length cDNAs were cloned into the chicken β-actin CMV (cytomegalo-virus) promoter driven expression vector pCAGGS (provided by J. L. R. Rubenstein): DsRed2 (Clontech, Cambridge, UK), Cxcl12 (IMAGE clone 3984285; Open Biosystems, Huntsville, AL).
Tamoxifen (Sigma) was dissolved in corn oil (Sigma) at 20 mg/ml. For temporal removal of Cxcr4, pregnant females with embryos at E15.5 were intraperitoneally injected with tamoxifen at 3 mg per 40 g animal.
Embryos older than E13.5 and postnatal mice were perfused with 1× PBS followed by 4% paraformaldehyde (PFA). Dissected brains were postfixed in 4% PFA overnight and then cryoprotected in 30% sucrose until they sank. Brains were frozen and sectioned coronally at 40 μm on a cryostat. Floating sections were stained with the following antibodies according to standard protocols: rabbit or mouse anti-GFP (1:1000; Invitrogen), rat anti-CTIP2 (1:1000; Abcam, Cambridge, MA), rabbit anti-calretinin (1:1000; Chemicon, Temecula, CA), rabbit anti-neuronal nitric oxide synthase (nNOS) (1:1000; Zymed, San Francisco, CA), mouse anti-parvalbumin (1:1000; Swant, Bellinzona, Switzerland), mouse anti-calbindin (1:1000; Swant), rabbit anti-NPY (1:1000; Immunostar, Hudson, WI). Primary antibodies were detected with secondary antibodies (goat anti-rabbit, goat anti-mouse, or goat anti-rat) conjugated to Alexa fluorochromes (1:1000; Invitrogen).
In situ hybridization
Embryos older than E13 were perfused with 4% PFA and brains were dissected and postfixed in 4% PFA for 4 h (for E13.5–E15.5 brains) or overnight (for brains older than E15.5). Fixed brains were cryoprotected in 30% sucrose before embedding in OCT. Brain tissues were cut on a cryostat at 20 μm and directly mounted onto Superfrost slides (Fisher Scientific, Houston, TX). All the slides were stored at −80°C before use. For in situ hybridization (ISH), slides were warmed to 55°C, fixed in 4% PFA for 30 min, treated with proteinase K (50 mg/ml) for 1.5 min, and fixed again with 4% PFA for 30 min. Acetylation was performed using 0.25% acetic anhydride in 0.1 m triethanolamine, pH 8.0, for 10 min, followed by three PBS washes. Slides were incubated with hybridization buffer [50% formamide, 5× SSC, 0.3 mg/ml yeast tRNA, 100 mg/ml heparin, 1× Denhart's, 0.1% Tween 20, 0.1% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate), 5 mm EDTA] for 30 min at 65°C, followed by overnight incubation with a digoxigenin-labeled probe. Three high-stringency washes were performed with 0.2× SSC at 65°C. Slides were then incubated with horseradish alkaline phosphatase (AP)-conjugated anti-digoxigenin and NBT (nitroblue tetrazolium)/BCIP (5-bromo-4-chloro-indolyl phosphate) for signal detection. The probes and their sources were as follows: Cxcl12 (IMAGE clone 3984285) and Cxcr4 (IMAGE clone 3592479; Open Biosystems), and Lhx6 and GAD67 (provided by J. L. R. Rubenstein).
Image analysis and quantification
Images were acquired using a cooled-CCD camera (QCapture Pro; QImaging, Burnaby, British Columbia, Canada). To quantitate the distribution of the recombined interneurons, counting boxes of equal size in controls (Z/EGTg/+;Dlx5/6CreTg/+;Cxcr4flox/+) and Dlx5/6-cKO (Z/EGTg/+;Dlx5/6CreTg/+;Cxcr4flox/−) animals were drawn in cingulate cortex, motor cortex, and somatosensory cortex at the bregma level of −0.94 mm. Six pairs of animals were analyzed. The numbers of GFP+ cells were normalized with the average values of the corresponding regions in the controls. For Lhx6-GFP+ animals with in utero electroporation of pCAG-DsRed2 alone, or together with pCAG-Cxcl12 targeted into the somatosensory cortex, counting boxes of the same size were drawn in somato-sensory cortex or cingulated cortex in the electroporated sides and contralateral sides. The ratio was calculated as the number of Lhx6-GFP+ cells in the electroporated side divided by the number of Lhx6-GFP+ cells in the contralateral side within the corresponding counting boxes. To further analyze the laminar distribution of Lhx6-GFP+ cells in the somatosensory cortices of the electroporated animals, a grid of 10 equal horizontal bins were drawn in the counting boxes. Bin 1 approximately corresponds to the marginal zone and bin 10 to the lower end of layer VI. The data were presented as the percentage of the total number within the boxes.
The results were expressed as the mean ± SEM for n given samples. Data were analyzed using two-tailed Student's t test with unequal variance. Any value of p ≤ 0.05 was considered significant.
Coronal brain slices (300 μm thick) were cut from 2- to 3-week-old mice on a Leica vibrating microtome in normal ice-cold artificial CSF. After 1–2 h of incubation at room temperature, slices were transferred to a submersion chamber on an upright Olympus (Tokyo, Japan) BX51 microscope, and layer 4 pyramidal cells in somatosensory “barrel,” or in a medial region (cingulate cortex), were visualized by infrared-differential interference contrast optics. The electroporated region of the slice was visualized by epifluorescence, and cells were targeted within the confines of the transfected area or in the equivalent region from the contralateral side. The extracellular solution contained the following (in mm): 119 NaCl, 2.5 KCl, 26 NaHCO3, 1 Na2PO4, 11 glucose, 2.5 CaCl2, and 1.3 MgCl2, and saturated with 95% O2/5% CO2. The intracellular solution contained the following (in mm): 135 CsMeSO4, 8 NaCl, 10 HEPES, 0.3 Na3GTP, 4 MgATP, 0.3 EGTA, and 5 QX-314 (lidocaine N-ethyl bromide). Experiments in which series resistance changed by >20% were excluded from analysis. Spontaneous IPSCs were collected at a holding potential of 0 mV. Under our conditions, the GABA reversal potential was approximately −45 mV, and thus the IPSCs were recorded as outward currents. Spontaneous events (75–125) were collected per cell and semiautomatically detected by in-house software in Igor Pro (WaveMetrics, Lake Oswego, OR). The Kolmogorov–Smirnov test was used to obtain p values for the analysis of cumulative distributions.