**3.1** *Cdh6* **gene expression along cortical layer IV neurons in the mouse primary somatosensory barrel field is regulated by an inter-genic region**

The cerebral cortex (also called the 'neocortex' or the 'isocortex') is a mammal-specific brain region with layered cellular organisation in its radial direction (Rakic, 1988). It can further be subdivided into functional areas in its tangential direction, with each area harbouring distinct layer components, constituting fundamental units for higher brain functions that are unique to mammals (O'Leary et al., 2007). How this characteristic brain region can emerge during development as well as through evolution has been one of the important research subjects in the field of neuroscience; however, little is known about the genetic cascade required to elaborate the intricate cytoarchitecture in the cerebral cortex. In this context, classic cadherins have very unique features in their expression patterns: each cadherin subclass shows a distinct cortical layer and/or area specificity at the perinatal stages in mouse and other mammalian species, such as ferrets and humans (Krishna-K et al., 2009; Suzuki et al., 1997; Wang et al., 2009). Classic cadherins might, therefore, provide ideal genetic clues in systematically understanding the molecular mechanism of cortical development.

In the previous studies, we have focused on mouse cadherin-6 (*Cdh6*), one of classic cadherin subclasses whose expression demarcates subsets of cortical layers and/or areas (Suzuki et al., 1997; Inoue et al., 1998), and we found out that a 58 kbp long 3 prime territory to mouse *Cdh6* gene (Segment X in Inoue et al., 2008a) is required for its mRNA expression along the cortical layer IV-barrel neurons in the primary somatosensory area (S1) at postnatal day 7 (P7; Inoue et al., 2008b; our unpublished data). In the present study, we sought to further narrow down the responsible territory for the expression by systematically deleting genomic regions from the reporter modified BAC clone that can recapitulate *Cdh6* expression in S1-barrel layer IV neurons at P7. To this end, we first referred to the evolutionary conserved region (ECR) browser in which conserved genomic regions among various species are aligned and annotated (http://ecrbrowser.dcode.org). Compared with human and dog genome sequences, as many as 60 ECRs – with more than 70% similarities in a window larger than 100 bp – were found in Segment X (data not shown), and we roughly divided Segment X into three regions (regions a~c in Figure 1) by means of ECR locations. We then sought to differentially delete two of the regions in Segment X from the reporter modified BAC clone *RP23-78N21* so as to effectively narrow down the responsible territory (Asami et al., 2011; Constructs #1 and #2 in Fig. 1). We subsequently obtained three

*PBS*: One litre of 10x PBS stock contains 80 g of NaCl, 2 g of KCl, 11.5 g of Na2HPO4 and 2 g of KH2PO4. We sterilised the stock solution by autoclaving and stored it at room temperature. *TAE*: One litre of 50x TAE solution contains 242 g of Tris-Cl, 57.1 ml of CH3COOH and 100

*TBE*: One litre of 10x TBE solution contains 108 g of Tris-Cl, 55 g of boric acid and 40 ml of

*Tyrode's solution*: Five litres of Tyrode's solution contains 40.0 g of NaCl, 1.0 g of KCl, 1.0 g of CaCl2, 1.05 g of MgCl2·6H2O, 0.285 g of NaH2PO4·2H2O, 5.0 g of NaHCO3 and 5.0 g of glucose. To avoid the possible precipitation of salts, we added these reagents in this order

**3.1** *Cdh6* **gene expression along cortical layer IV neurons in the mouse primary** 

The cerebral cortex (also called the 'neocortex' or the 'isocortex') is a mammal-specific brain region with layered cellular organisation in its radial direction (Rakic, 1988). It can further be subdivided into functional areas in its tangential direction, with each area harbouring distinct layer components, constituting fundamental units for higher brain functions that are unique to mammals (O'Leary et al., 2007). How this characteristic brain region can emerge during development as well as through evolution has been one of the important research subjects in the field of neuroscience; however, little is known about the genetic cascade required to elaborate the intricate cytoarchitecture in the cerebral cortex. In this context, classic cadherins have very unique features in their expression patterns: each cadherin subclass shows a distinct cortical layer and/or area specificity at the perinatal stages in mouse and other mammalian species, such as ferrets and humans (Krishna-K et al., 2009; Suzuki et al., 1997; Wang et al., 2009). Classic cadherins might, therefore, provide ideal genetic clues in systematically

In the previous studies, we have focused on mouse cadherin-6 (*Cdh6*), one of classic cadherin subclasses whose expression demarcates subsets of cortical layers and/or areas (Suzuki et al., 1997; Inoue et al., 1998), and we found out that a 58 kbp long 3 prime territory to mouse *Cdh6* gene (Segment X in Inoue et al., 2008a) is required for its mRNA expression along the cortical layer IV-barrel neurons in the primary somatosensory area (S1) at postnatal day 7 (P7; Inoue et al., 2008b; our unpublished data). In the present study, we sought to further narrow down the responsible territory for the expression by systematically deleting genomic regions from the reporter modified BAC clone that can recapitulate *Cdh6* expression in S1-barrel layer IV neurons at P7. To this end, we first referred to the evolutionary conserved region (ECR) browser in which conserved genomic regions among various species are aligned and annotated (http://ecrbrowser.dcode.org). Compared with human and dog genome sequences, as many as 60 ECRs – with more than 70% similarities in a window larger than 100 bp – were found in Segment X (data not shown), and we roughly divided Segment X into three regions (regions a~c in Figure 1) by means of ECR locations. We then sought to differentially delete two of the regions in Segment X from the reporter modified BAC clone *RP23-78N21* so as to effectively narrow down the responsible territory (Asami et al., 2011; Constructs #1 and #2 in Fig. 1). We subsequently obtained three

**somatosensory barrel field is regulated by an inter-genic region** 

understanding the molecular mechanism of cortical development.

ml of 0.5M EDTA (pH8.0).

and sterilised by filtration and stored at 4C.

**3. Results and discussions** 

0.5M EDTA (pH8.0).

Fig. 1. An intergenic segment of mouse *Cdh6* is found to be necessary for the postnatal barrel area specific expression in the cerebral cortex.

The uppermost part of the figure indicates the genomic structure of mouse *Cdh6*, with its exons being designated by short vertical lines. ATG, translation start site; TSS, transcription start site. A BAC clone *RP23-78N21* is initially modified to harbour a beta-galactosidase gene cassette and a SV40 polyadenylation signal (*LacZpA)* in a frame to the *Cdh6* gene via homologous recombination (Rec) in a recombinogenic bacterial strain. This original BAC is further engineered by homologous recombination so as to generate deletion constructs #1~#3. Note that transgenic (Tg) mice with original BACs and construct #2 strongly recapitulate the somatosensory barrel-specific expressions (black arrow heads), while those with constructs #1 and #3 do not yield the expression at the postnatal day 7 (P7; white arrow heads). From these results, a 5840-bp territory (orange box) is determined to be a critical regulatory region for the barrel-specific expression of *Cdh6* at P7. At the upper left corner of each panel, the ratio of brain samples exhibiting reproducible reporter expression over the total number of independent transgenic mouse lines generated is noted.

stable, independent BAC transgenic (Tg) mouse founders from Construct #1 and five from Construct #2. In the former founders, none of them recapitulated the reporter expression in S1-barrel layer IV neurons (Fig. 1, white arrow heads), which are strongly marked by the original BAC trans-genesis (Fig. 1, black arrow heads). In contrast, four out of five founders reproduced the intense expression profile of S1-barrel layer IV neurons, while one of them showed no reporter expression in the brain (probably due to the positional effect of the BACs' integration site). From these results, it is strongly suggested that the most 5 prime third of Segment X (region a in Fig. 1) is responsible for *Cdh6* expression in the S1-barrel layer IV neurons at P7.

In order to further to narrow down the responsible territory, we designed the Construct #3 in which a fragment containing the most 3 prime third of region a is excluded from the original BACs. Among seven Tg founders generated from Construct #3, we could not observe the intense reporter expression of S1-barrel layer IV neurons at all (white arrow heads in Fig. 1), while we found that neurons in the other cortical layers (i.e. layers II/III) and/or areas at P7 maintained their conspicuous expression compared to the original *Cdh6*- BAC-Tg lines (Fig. 1). Taken together, we concluded that a 5,884 bp territory containing 11 ECRs is required for *Cdh6* expression in S1-barrel layer IV neurons at P7.

To our knowledge, this is the first time that a distinct gene regulatory fragment for a defined layer and/or area has been observed, suggesting that separable genetic programs may serve the pattering of each cortical layer and/or area during development. The further characterisation of the gene regulatory elements that directly interact with the 5,884 bp territory would be an important next step in understanding how the cortical layer and/or area identity is strictly determined during development. In this connection, it is noticeable that the 5,884 bp territory contains many of the transcription factor binding motifs, such as *RORbeta*, whose expression is already known to be restricted to defined sets of cortical layers and/or areas (Dye et al., 2011; Hirokawa et al., 2008; Nakagawa & O'leary, 2003). Since recent reports suggest that *RORbeta* harbours an instructive role in elaborating barrel cytoarchitecture and/or circuitries (Jabaudon et al., 2011), it would be of great interest to rigorously evaluate how these transcription factors are involved in establishing *Cdh6* expression along S1-barrel layer IV neurons at P7 which might have functional significance in driving cell segregations to form and/or maintain the barrel cytoarchitecture.
