**3.2 Retinoic acid (RA) related cell signalling machineries might be involved in the cortical barrel patterning**

RA signalling regulates many of the morphogenetic events at the early embryonic stage, such as A-P axis formation, the establishment of L-R asymmetry and so on (Kiecker & Lumsden, 2005; Niederreither & Dolle, 2008). It has recently been suggested that RA might also have physiological roles in cortical development at the later embryonic stages (Siegenthaler et al., 2006; Smith et al., 2001). We thus next tried to examine the possible roles of RA related cell signalling machinery in the cortical area pattering during development. By using the same BAC clone *RP23-78N21*, we replaced the *Cdh6*-ATG exon to an expression cassette for the membrane-bound form of enhanced green fluorescent protein (GAP43-EGFP) by means of homologous recombination and generated transgenic

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

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

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

**3.2 Retinoic acid (RA) related cell signalling machineries might be involved in the** 

RA signalling regulates many of the morphogenetic events at the early embryonic stage, such as A-P axis formation, the establishment of L-R asymmetry and so on (Kiecker & Lumsden, 2005; Niederreither & Dolle, 2008). It has recently been suggested that RA might also have physiological roles in cortical development at the later embryonic stages (Siegenthaler et al., 2006; Smith et al., 2001). We thus next tried to examine the possible roles of RA related cell signalling machinery in the cortical area pattering during development. By using the same BAC clone *RP23-78N21*, we replaced the *Cdh6*-ATG exon to an expression cassette for the membrane-bound form of enhanced green fluorescent protein (GAP43-EGFP) by means of homologous recombination and generated transgenic

ECRs is required for *Cdh6* expression in S1-barrel layer IV neurons at P7.

layer IV neurons at P7.

cytoarchitecture.

**cortical barrel patterning** 

Fig. 2. Possible involvement of retinoic acid (RA) related cell signalling machineries in the cortical barrel patterning.

A BAC clone *RP23-78N21* is modified to harbour a membrane-bound form of EGFP cassette and SV40 polyadenylation signal (GAP43-EGFP-pA) in frame to the *Cdh6* gene via homologous recombination (Rec) in a recombinogenic bacterial strain (upper most part of the figure). (A-D) A Tg mouse with EGFP modified BACs recapitulated *Cdh6* expression at P7. OB, olfactory bulb; S1 primary somatosensory area. The boxed area d in panel C is magnified in panel D. Note that Layer IV barrels in S1 are illuminated in this Tg mouse line. (E-H) Effects of RA on barrel development. RA is intraperitoneally injected into the mother (E, F) or the pups (G, H) and the EGFP expression profile is evaluated at P7. Note that this is only the case with E14.5 injections, which affect the formation of barrel cytoarchitecture, highlighting the role of RA in early neocortical development.

mouse founders (Inoue et al., 2009). In the Tg cerebral cortex, we found that GFP expression shows exactly the same patterns as with the original LacZ-Tg mice (Fig 1 and 2). In particular, this GFP-BAC-Tg mouse line illuminated the S1 barrel structure of the whole mount brain preparations, allowing us to easily image the S1-barrel territory whose identification generally requires specific histological staining processes, such as the CO staining method.

We then administrated RA to the mothers or pups for GFP-BAC-Tg, with the concentration reported to induce abnormality in the cortex (Smith et al., 2001) and evaluated how RA affects the GFP expression patterns at P7. As a result of this, no drastic change was observed for the cortical barrel patterning when RA was administrated later than embryonic day 17.5 (E17.5). However, RA administration at E14.5 resulted in massive perturbation of S1 barrel patterning, with ambiguous area boundaries illuminated by GFP expression. Noticeably, the intensity of the GFP expression appeared to be decreased due to the qualitative and quantitative differences among *Cdh6*::*GFP* positive cortical cells and/or thalamocortical axon terminals. These results imply that the role of RA in regulating *Cdh6* expression and/or cortical area pattering is just limited to those embryonic stages earlier than E14.5.

It is now widely accepted that cortical area patterning begins as early as mouse E12.5 when the counter-gradient of the transcription factors Pax6 and Emx2 is established in the cortical ventricular zones (Bishop et al., 2000; Hamasaki et al., 2004). This gradation pattern, as generated by such secreted molecules as *Fgf8*, is shown to be the basis of cortical arealisation yet other transcriptional factors, such as *Coup-TFI,* could regulate the area-specific differentiation of distinct subtypes of cortical neurons independently of *Fgf8*-*Pax6*/*Emx*2 gene functions (Armentano et al., 2007; Fukuchi-Shimogori & Grove, 2001, 2003). Our results, together with a previous series of studies, thus suggest that RA accumulated earlier than E14.5 might play a role in cortical arealisation by affecting the production, migration, positioning and/or circuit formation of the cortical S1 barrel layer IV neurons that eventually express *Cdh6*. The next critical step would be to examine whether RA-related signalling could be interactive with the 5,884 bp territory identified in this study that contains *RORbeta* related transcription factor binding motifs.
