zones in barrel cortex including our DZ defined here does not receive inputs from the primary somatosensory thalamic nucleus; instead it receives inputs from a higher order thalamic nucleus and adjacent primary somatosensory areas as well [45–47], [59], [60]. The high PC1 score is consistent with this observation and may support a proposal that the dysgranular non-barrel cortex is a higher order somatosensory area [47]. Finally, PC1 has contribu- tions from both RORbeta and ER81 layer maps, but in the reverse direction. These genes are enriched in layers 4 and 5, which are generally considered as the input and output layers [61]. We speculate that the maturation of the structures of layers 4 and 5 is coordinated so that the primary sensory areas are specialized for input reception. Compared with the interpretation of PC1, that of PC2 is more difficult. We could think of possible developmental causes for the conspicuous lateral-to-medial gradient of PC2: it may reflect cortical patterning by a gradient of regulatory genes [58], [62], or a cortical migratory stream [63]. However, the functional significance of such a gradient in the mature neocortex remains unclear. Arimatsu and coworkers report that Nurr1-positive neurons send corticocortical but not corticothalamic projections in the rat cortex [19]. We found that this projection specificity is also conserved in monkeys [23]. These observations suggest that PC2 may represent a special type of cortico-cortical connectivity. It is quite intriguing that Nurr1 mRNA is expressed by a subtype of neurons distinct from those expressing RORbeta or ER81 mRNAs despite the extensive intermingling (Fig. 3). The negative correlations of Nurr1 with RORbeta or ER81 contribu- tion in PC1 and PC2 (Fig. 7A) raise the possibility that the same organizing principle differentially affects different cell types. We predict that there may be some rules that coordinate the expression of thousands of genes in organizing the neocortical structure. How such coordination occurs is, at present, an open question. The double ISH and cortical box method are useful tools for analyzing such rules and for revealing the principles behind them. Materials and Methods Animals and tissue preparation Four adult male Sprague Dawley rats (one for double ISH, three for cortical box method of single ISH) were purchased from Japan SLC, Inc. (Hamamatsu, Japan) and perfused through the heart with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) under deep anesthesia induced by Nembutal (50 mg/kg body weight, i.p.). All the experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Institute of Health (USA) publication number 86–23, 1985) and the guidelines of the Okazaki National Research Table 1. Primers used to clone RORbeta gene segments. Plasmid name mRORbeta rRORbeta2 rRORbeta3 doi:10.1371/journal.pone.0003266.t001 Primer set GTGTACAGCAGCAGCATTAGCA GGTCTCATCATCCAGGTGRTTC AAAGCAAGCACATTGGAGAG GTCAATGACGTGCCCGTTGG AACAAACAGAAGAGCCCCAC GCCAACGGGCACGTCATTGACC ISH Profiling of Rat Cortex Institutes in Japan. We made all efforts to minimize the number of animals used and their suffering. Probe preparation The cDNA fragments of mouse or rat RORbeta were obtained by polymerase chain reaction (PCR) using the primers listed in Table 1 and subcloned into the pBlueScriptII vector. Each of the three probes listed in Table 1 exhibited very similar expression patterns for both mouse and rat brains (data not shown), validating the reproducibility of the ISH result. To obtain the data presented in this paper, we used two probes, rRORbeta2 and rRORbeta3, mixed together for hybridization. The probes for the ER81 and Nurr1 gene were previously described [21]. The digoxygenin (DIG)- and fluorescein (FITC)-labeled riboprobes were produced by in vitro transcription using these plasmids as templates. The riboprobes were purified using ProbeQuant 50 spin column (Amersham Biosciences, Little Chalfont, UK). Single-color ISH A silicon template was used to cut vertically the brain into half (anterior and posterior parts). Coronal sections from both hemispheres were cut at 40 mm thickness using a freezing microtome. Special care was taken to maintain the orientation of sections. Every seventh section was preserved for ISH using RORbeta, ER81 and Nurr1 gene probes and Nissl staining. The remaining sections were frozen for later use. These sections were equivalent to the entire hemisphere with an approximately 280 mm interval. ISH was carried out as previously described [21], [64]. Briefly, free-floating sections were treated with proteinase K (1 mg/mL) for 30 minutes at 37uC, acetylated, then incubated in a hybridization buffer containing 0.25–0.5 mg/mL digoxigenin-labeled riboprobes at 60uC. The sections were sequentially treated in 26 standard saline citrate (SSC)/50% formamide/0.1% N-lauroylsarcosine for 20 minutes at 60uC, twice; 30 minutes at 37uC in RNase buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 500 mM NaCl) containing 20 mg/mL RNase A (Sigma-Aldrich, St. Louis, MO); 20 minutes at 37uC in 26 SSC/0.1% N-lauroylsarcosine, twice; 20 minutes at 37uC in 0.26 SSC/0.1% N-lauroylsarcosine, twice. The hybridized probe was detected with an alkaline phosphatase-conjugated anti-DIG antibody using a DIG nucleic acid detection kit (Roche Diagnostics, Indianapolis, IN, USA). There were no apparent signals in control sections examined with the sense probes. ISH causes considerable tissue section shrinkage (80%). However, the scale bars in the figures are not adjusted for such shrinkage. Double ISH was carried out using DIG- and FITC-labeled riboprobes as previously described [23]. The sections were cut to 15–20 mm thickness. The hybridization and washing were carried out as described above, except that both DIG and FITC probes template Mouse brain cDNA Rat brain cDNA Rat brain cDNA Target sequence (CDS = +1 to +862) From 2136 to +676 From 2840 to 293 From 273 to +1029 PLoS ONE | www.plosone.org 10 September 2008 | Volume 3 | Issue 9 | e3266