Abstract

Cadherins are superfamily of Ca2+-dependent transmembrane glycoproteins with more than 100 members. They play a role in a wide variety of developmental mechanisms, including cell proliferation, cell differentiation, cell–cell recognition, neurite outgrowth and synaptogenesis. We cloned 16 novel members of the classic cadherin and δ-protocadherin subgroups from ferret brain. Their expression patterns were investigated by in situ hybridization in the developing primary visual cortex (V1) of the ferret. Fifteen out of the 16 cadherins are expressed in a spatiotemporally restricted fashion throughout development. Each layer of V1 can be characterized by the combinatorial expression of a subset of cadherins at any given developmental stage. A few cadherins are expressed by subsets of neurons in specific layers or by neurons dispersed throughout all cortical layers. Generally, the expression of protocadherins is more widespread, whereas that of classic cadherins is more restricted to specific layers. At the V1/V2 boundary, changes in layer-specific cadherin expression are observed. In conclusion, our results suggest that cadherins provide a code of potentially adhesive cues for layer formation in ferret V1. The persistence of expression in the adult suggests a functional role also in the mature cortex.

Introduction

One of the most salient anatomical features of the mammalian neocortex is its organization into 6 layers. Most cortical cells are born in the ventricular and subventricular zones of the proliferative neuroepithelial layer. A fraction of the neuroepithelial cells leaves the mitotic cycle and differentiates into early neurons, which are guided to migrate pialward by radial glial fibers. The earliest born neurons form the preplate. Later-born neurons migrate into the preplate to form the cortical plate that splits the preplate into the marginal zone (MZ) (prospective layer I) and the subplate. As more neurons arrive in the cortical plate, the 6 neocortical layers are formed in an inside-out fashion (Angevine and Sidman 1961; Rakic 1974; Caviness et al. 1995; Rakic and Caviness 1995). In addition, the cortical plate becomes populated by interneurons that are born in the ganglionic eminences and migrate tangentially into neocortex (Anderson et al. 1997; Nadarajah and Parnavelas 2002).

The molecular events that regulate the interaction of the migrating cortical cells with their environment as well as their final positioning within the cortical plate are beginning to be understood. The differences in neuronal composition, developmental timing and connectivity across cortical layers strongly suggest the existence of a vast number of genes with layer- and region-specific patterns of expression. Consistent with this idea, several gene regulatory proteins and morphogenetic molecules, which are expressed in specific layers, were identified. These molecules include Sidekicks, EphrinA5, OTX1, CUTL2, CALB1, N-cadherin, R-cadherin, PCDH8, Reelin, LAMB1, NR2E1, NR2F2, VIP, CNR1, and LIX1 (Rakic 1988; Obst-Pernberg et al. 2001; Hevner et al. 2003; Rash and Grove 2006; Hevner 2007; Molyneaux et al. 2007; Watakabe et al. 2007; Zhou et al. 2007).

In an attempt to identify additional markers for cortical layers and regions, we focused on cadherins in the present study. Cadherins are a large family of Ca2+-dependent cell adhesion glycoproteins, with more than 100 members in vertebrates. Cadherins mediate cell–cell adhesion and signal transduction and are grouped into subfamilies that are designated as classic cadherins, desmosomal cadherins, protocadherins, Flamingo cadherins and FAT molecules (for reviews, see Nollet et al. 2000; Frank and Kemler 2002; Hirano et al. 2003). Among these subfamilies, protocadherins are the largest one and contain several subgroups, such as α-, β-, and γ-protocadherins that form one large cluster of genes in both the mouse and human genome. More recently, a subgroup of nonclustered protocadherins was recognized and classified as δ-protocadherins (Redies et al. 2005; Vanhalst et al. 2005).

The vast majority of cadherins is expressed in distinct patterns in the developing and mature central nervous system of vertebrates, where they play multiple roles in the segregation of neuronal precursor populations, neurite outgrowth, axon guidance and synapse formation (for reviews, see Redies 1997, 2000; Hirano et al. 2003; Takeichi 2007). Expression analysis and functional studies have led to the idea that the homotypic adhesions mediated by cadherins may provide an adhesive code for the selective association of neuronal structures during the functional differentiation of the nervous system (Redies et al. 1993; for reviews, see Redies and Takeichi 1996; Redies 2000). Therefore, studying the expression of cadherins can help us to understand the molecular cues regulating corticogenesis.

Several cadherins have been mapped in the rodent forebrain, for example, Rcad (Cdh4) and Ncad (Cdh2; Redies and Takeichi 1993; Obst-Pernberg et al. 2001), Cdh6, Cdh8, and Cdh11 (Korematsu and Redies 1997; Suzuki et al. 1997), α-protocadherins (Kohmura et al. 1998; Zou et al. 2007), γ-protocadherins (Zou et al. 2007), and δ-protocadherins (Hirano et al. 1999; Redies et al. 2005; Vanhalst et al. 2005; Gaitan and Bouchard 2006; Kim et al. 2007; Hertel et al. 2008). Each of these cadherins shows a spatially restricted expression in a specific subset of gray matter structures. Most cadherins are expressed also in a layer-specific fashion in the developing cortex. Typically, the layer-specific distribution of cadherins changes from region to region within cortex. Some cadherins have been used as markers for cortical regions in genetically altered mice (Cdh6, Cdh8, and Cdh11; Miyashita-Lin et al. 1999; Nakagawa et al. 1999; Rubenstein et al. 1999; Bishop et al. 2000).

All these previous studies on cadherin expression in cerebral cortex have focused on single or a few cadherins, often only at a specific stage of cortical development and in a particular cortical area. Here, we map systematically the expression of fifteen novel classic cadherins and δ-protocadherins by in situ hybridization in the developing primary visual cortex (V1) of the ferret, which serves as a model system for cortical development (Rockland 1985). Ferrets have a relatively short gestational period (41–42 days), coupled with a protracted period (35 days) of postnatal cortical neurogenesis. Unlike the mouse, the ferret has a large cerebral cortex and the duration of neuron production permits a high temporal resolution of developmental events and stages of growth (McSherry 1984; Jackson et al. 1989).

Materials and Methods

Animals and Preparation of Tissues

Ferrets bred in captivity were obtained from the Federal Institute of Risk Research in Berlin-Marienfelde, Germany. All animals used in this study were deeply anesthetized by an overdose of intraperitoneal pentobarbital followed by decapitation, according to institutional and national guidelines on the welfare of animals. Embryos were removed from timed pregnant ferrets at 23 days after conception (E23), at E30 and at E38. Postnatally, brains from the following stages were obtained: postnatal day 2 (P2), P13, P25, P33, P46, P60 and adult. The day of birth was designated as P0. The number of animals used in this study was kept at a minimum and efforts were made to minimize animal suffering.

For embryonic stages, the skull above the brain was opened for better diffusion of the fixative. For postnatal stages, brains were removed from the skull. Specimens from brains up to P33 were fixed by immersion in ice-cold 4% formaldehyde solution in phosphate-buffered salt solution (PBS; 13 mM NaCl, 7 mM Na2HPO4, 3 mM NaH2PO4; pH 7.4) and frozen in TissueTek compound (Science Services, Munich, Germany). Brains from P46 animals and older were flash frozen in 2-methyl-butane chilled to about −40 °C by adding dry ice. All specimens were stored at −80 °C until sectioning.

Parasagittal and transverse sections of 20 μm thickness were cut in a cryostat (HM 560 Cryo-Star Cryostat, Microm International, Walldorf, Germany) and thawed directly onto SuperFrost plus slide glasses (Menzel, Braunschweig, Germany). The sections were dried at 50–56 °C. Totally, 36 entire brains or isolated occipital lobes were cut with each specimen yielding about 160–200 sections.

RNA Isolation and cDNA Synthesis

Brains of E38 pups or adult ferrets were flash frozen in liquid nitrogen. Total RNA was isolated using the RNeasy protect mini kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The purity of the RNA samples was assessed spectrophotometrically and total RNA was quantified from the absorbance at 260 nm. First-strand complementary DNA was synthesized from the ferret total RNA using SuperScript III First-Strand Synthesis SuperMix (Invitrogen, CA) according to the manufacturer's protocol in a thermocycler (Mastercycler personal, Eppendorf, Hamburg, Germany).

PCR Amplification

Several classic cadherins and all known δ-PCDH molecules were amplified by PCR using both specific and degenerate primers designed for regions, which are highly conserved between mouse, rat, dog, and human for different cadherin molecules. Degeneracy levels up to 16 were used. Degenerate primers were designed manually by using the ClustalX multiple alignment tool and verified by the CODEHOP online primer designing software tool. The degeneracy levels of type 2 classic cadherin primers (Price et al. 2002) were 192-fold. The primers are listed in Table 1.

Table 1

Primers used to obtain cadherin fragments by RT-PCR

Cadherin Primer sequence Size (bp) Accession number 
CDH4 5′ AAG CGT GAC TGG GTC ATC C 1501 EU665238 
 3′ TTG AGG ATC TTT TCG C   
CDH6 5′ TGG (AG)T(AGCT) TGG AA(CT) CA(AG) (AT)T(GCT) 1824 EU665239 
 3′ CC(AGCT) CC(AGCT) CC(CT) TC(AG) TC(AG) T(CT)(AG) TA   
CDH7 5′ TGG (AG)T(AGCT) TGG AA(CT) CA(AG) (AT)T(GCT) 1824 EU665240 
 3′ CC(AGCT) CC(AGCT) CC(CT) TC(AG) TC(AG) T(CT)(AG) TA   
CDH8 5′ TGG (AG)T(AGCT) TGG AA(CT) CA(AG) (AT)T(GCT) 1825 EU665241 
 3′ CC(AGCT) CC(AGCT) CC(CT) TC(AG) TC(AG) T(CT)(AG) TA   
CDH11 5′ CCT GAC CCT GTG CTC GTG 1110 EU665242 
 3′ ACC GTC CTC TGG ATT GAT AGT G   
CDH14 5′ TGG (AG)T(AGCT) TGG AA(CT) CA(AG) (AT)T(GCT) 1474 EU665243 
 3′ CC(AGCT) CC(AGCT) CC(CT) TC(AG) TC(AG) T(CT)(AG) TA   
CDH20 5′ TGG (AG)T(AGCT) TGG AA(CT) CA(AG) (AT)T(GCT) 2000 EU665244 
 3′ CC(AGCT) CC(AGCT) CC(CT) TC(AG) TC(AG) T(CT)(AG) TA   
PCDH1 5′ GAC CTC ACC ATC AAG GT 1961 EU665245 
 3′ TGG GGG CAT ACA GGT CC   
PCDH7 5′ TGA TCG TGA AGG GGG CGC TGG ACC G 2326 EU665246 
 3′ CCT GCT CCC ACA AAT GTG TTG GCT GG   
PCDH8 5′ TTY AGY CTY TGC TGG GTG CTC TC 1702 EU665247 
 3′ GY TGG CGC AGM GTC TCA TAG TC   
PCDH9 5′ CTG GTG CTA CCA GAT GCA TGG C 1749 EU665248 
 3′ CCT CTT GTC CGG AGA GGC CTG G   
PCDH10 5′ GGA GAT CGA IGT GCT GGA 1860 EU665249 
 3′ CCG CCC TGG GGC TCC ACG   
PCDH11 5′ CAT GCC ACA GAT GCT GAC ATA GGT G 2044 EU665250 
 3′ GCA ACC AKG ATC TTG ACA TAG TCA C   
PCDH17 5′ CGA CGG CAC CAA GTT CCC 2038 EU665251 
 3′ CCC ATG TAA TTG GGC TCT G   
PCDH18 5′ GCA GCA GTT GGG ACT CG 2351 EU665252 
 3′ GGC ATC CAG CAC TGG TCA GAG   
PCDH19 5′ AGC GCG CCG GGA CGG TGA TCG C 2896 EU665253 
 3′ GGG CTG CAG ATG GTC ACA TCG ACA G   
Cadherin Primer sequence Size (bp) Accession number 
CDH4 5′ AAG CGT GAC TGG GTC ATC C 1501 EU665238 
 3′ TTG AGG ATC TTT TCG C   
CDH6 5′ TGG (AG)T(AGCT) TGG AA(CT) CA(AG) (AT)T(GCT) 1824 EU665239 
 3′ CC(AGCT) CC(AGCT) CC(CT) TC(AG) TC(AG) T(CT)(AG) TA   
CDH7 5′ TGG (AG)T(AGCT) TGG AA(CT) CA(AG) (AT)T(GCT) 1824 EU665240 
 3′ CC(AGCT) CC(AGCT) CC(CT) TC(AG) TC(AG) T(CT)(AG) TA   
CDH8 5′ TGG (AG)T(AGCT) TGG AA(CT) CA(AG) (AT)T(GCT) 1825 EU665241 
 3′ CC(AGCT) CC(AGCT) CC(CT) TC(AG) TC(AG) T(CT)(AG) TA   
CDH11 5′ CCT GAC CCT GTG CTC GTG 1110 EU665242 
 3′ ACC GTC CTC TGG ATT GAT AGT G   
CDH14 5′ TGG (AG)T(AGCT) TGG AA(CT) CA(AG) (AT)T(GCT) 1474 EU665243 
 3′ CC(AGCT) CC(AGCT) CC(CT) TC(AG) TC(AG) T(CT)(AG) TA   
CDH20 5′ TGG (AG)T(AGCT) TGG AA(CT) CA(AG) (AT)T(GCT) 2000 EU665244 
 3′ CC(AGCT) CC(AGCT) CC(CT) TC(AG) TC(AG) T(CT)(AG) TA   
PCDH1 5′ GAC CTC ACC ATC AAG GT 1961 EU665245 
 3′ TGG GGG CAT ACA GGT CC   
PCDH7 5′ TGA TCG TGA AGG GGG CGC TGG ACC G 2326 EU665246 
 3′ CCT GCT CCC ACA AAT GTG TTG GCT GG   
PCDH8 5′ TTY AGY CTY TGC TGG GTG CTC TC 1702 EU665247 
 3′ GY TGG CGC AGM GTC TCA TAG TC   
PCDH9 5′ CTG GTG CTA CCA GAT GCA TGG C 1749 EU665248 
 3′ CCT CTT GTC CGG AGA GGC CTG G   
PCDH10 5′ GGA GAT CGA IGT GCT GGA 1860 EU665249 
 3′ CCG CCC TGG GGC TCC ACG   
PCDH11 5′ CAT GCC ACA GAT GCT GAC ATA GGT G 2044 EU665250 
 3′ GCA ACC AKG ATC TTG ACA TAG TCA C   
PCDH17 5′ CGA CGG CAC CAA GTT CCC 2038 EU665251 
 3′ CCC ATG TAA TTG GGC TCT G   
PCDH18 5′ GCA GCA GTT GGG ACT CG 2351 EU665252 
 3′ GGC ATC CAG CAC TGG TCA GAG   
PCDH19 5′ AGC GCG CCG GGA CGG TGA TCG C 2896 EU665253 
 3′ GGG CTG CAG ATG GTC ACA TCG ACA G   

For amplification, the REDTaq ReadyMix PCR system (Sigma-Aldrich, St Louis, MO) was used in a gradient thermocycler (Mastercycler, Eppendorf). Each amplification commenced with an initial denaturation step at 95 °C for 2 min followed by 35 cycles of denaturation at 94 °C for 3 min, primer annealing for 50 s at temperatures ranging from 54–60 °C, depending upon the primers, and extension at 72 °C for 2 min. Finally, extension was performed at 72 °C for 10 min to complete the synthesis of all strands. The PCR products were analyzed by electrophoresis in 1.2% agarose gels stained with ethidium bromide, and bands were visualized and photographed under ultraviolet light excitation (BioDoc Analyzer, Biometra, Germany). The size of the different cadherin fragments varied from 1.1 to 3 kb (Table 1).

Cloning of Cadherins

The resulting PCR products were purified (QIAquick gel extraction kit, Qiagen) and ligated into the pCR II-TOPO vector (TOPO TA Cloning Kit, Invitrogen) in accordance with the manufacturer's instructions. Several clones were picked after transformation of chemically competent Escherichia coli TOP 10F cells (Invitrogen) based on blue-white colony selection. The isolated plasmids (Qiaprep miniprep kit, Qiagen) were checked by restriction digestion to select a single plasmid harboring the desired sequence.

DNA Sequencing

All the inserts were sequenced using the SequiTherm EXCEL II DNA Sequencing Kit (Epicentre Biotechnologies, Madison, WI) according to the manufacturer's protocol in a DNA sequencer (LI-COR Biotechnology, Lincoln, NE). All confirmed cadherin molecules were sequenced again by a commercial company (MWG-Biotech, Ebersberg, Germany) using M13 forward, reverse and specific internal primers. The sequences have been submitted to the NCBI GenBank database. The accession numbers are listed in Table 1.

cRNA Probe Synthesis

Nonradioactive cRNA probes were produced for all the cadherin molecules with the digoxigenin (DIG) RNA Labeling Kit or the Fluorescein-RNA Labeling Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. The linearized plasmids were transcribed with T7 or SP6 RNA polymerase (New England Biolabs, Ipswich, MA) followed by labeling with digoxigenin or fluorescein to generate sense and antisense probes. Probes were purified by LiCl/EtOH precipitation or by using Quick Spin Columns (Roche Diagnostics). Correct probe size was verified by formaldehyde-agarose gel electrophoresis.

In Situ Hybridization

In situ hybridization was performed as described previously (Redies et al. 1993). Cryostat sections of 18- or 20-μm thickness were (post-) fixed with 4% formaldehyde in PBS and were pretreated with proteinase K and acetic anhydride. Sections were hybridized with cRNA probes at a concentration of about 1 ng/μL overnight at 70 °C in hybridization solution (50% formamide, 10 mM ethylene diaminetetraacetic acid [EDTA], 3× saline-sodium citrate (SSC), 1× Denhardt's solution, 10× dextran sulfate, 42 μg/mL yeast RNA, and 42 μg/mL salmon sperm DNA). After the sections were washed, alkaline phosphatase-coupled anti-digoxigenin Fab fragments were applied. For visualization of the labeled cRNAs, the sections were incubated with a substrate mixture of 0.03% nitroblue tetrazolium salt and 0.02% 5-bromo-4-chloro-3-indolyl phosphate for 1–3 days at room temperature or at 4 °C, until enough reaction product had formed. The sections were viewed and photographed under a microscope (BX40, Olympus, Hamburg, Germany) equipped with a digital camera (DP70, Olympus). Digitized images from in situ hybridization were adjusted in contrast and brightness with the Photoshop software (Adobe Systems, Mountain View, CA).

Tyramide Signal-Amplified Double-Fluorescent in Situ Hybridization

The in situ hybridization protocol was modified in the pre- and posthybridization steps by introducing a fluorescent reaction product by CARD in order to visualize the expression of more than one cadherin in a single section. Endogenous peroxidase activity was quenched with 1% hydrogen peroxide. Then, sections were hybridized with a mixture of both DIG- and fluorescein-labeled cRNA probes at a concentration of about 1 ng/μl each, overnight at 70 °C in hybridization solution (50% formamide, 10 mM EDTA, 3× SSC, 1× Denhardt's solution, 10× dextran sulfate, 42 μg/mL yeast RNA, and 42 μg/mL salmon sperm DNA). After the hybridized sections were washed, horseradish peroxidase (HRP)–coupled anti-digoxigenin Fab fragments were applied to bind to the digoxigenin-labeled cRNA probes. Enzymatic activity was visualized by incubating sections with a substrate solution containing the tyramide-coupled Alexa fluorophore A488 (Invitrogen) and H2O2, for 1 h at room temperature. Subsequently, for the detection of the fluorescein-labeled probe, HRP-coupled anti-fluorescein Fab fragments and tyramide-coupled Alexa fluorophore A568 were used. The sections were viewed and photographed under a confocal laser scanning microscope (SP5, Leica Microsystems, Wetzlar, Germany).

Results

Out of the 16 cadherins studied in the present work, all except PCDH18 are expressed in the primary visual cortex of the adult ferret. Each cadherin shows a distinct and layer-specific expression profile. In general, classic cadherins show a more restricted expression pattern than δ-protocadherins. Each cortical layer is thus marked by the combinatorial expression of multiple cadherins. However, not all cells in a given layer express the same combination of cadherins. Rather, in some layers, a given cadherin may be expressed only by a subset of cells.

Expression of the different cadherins begins at different times during development. Some cadherins are already expressed by the cells in the preplate (for example, CDH4, CDH11, CDH14, PCDH7, PCDH9, and PCDH10) or by the ventricular zone (e.g., CDH4, CDH6, CDH20, and PCDH1) at the earliest stage examined (E23). Most cadherins (for example, CDH8, CDH11, CDH20, PCDH1, PCDH7, PCDH9, PCDH10, PCDH11, PCDH17, and PCDH19) are expressed in the subventricular zone, the intermediate zone or the subplate before birth. All fifteen cadherins are expressed in the cortical plate (cortical layers II–VI) at birth. Layer I contains cells positive for a subset of cadherins (e.g., CDH4, CDH6, CDH7, CDH11, CDH14, CDH20, PCDH1, PCDH7, PCDH8, PCDH9, and PCDH10) in the mature visual cortex. During postnatal development, expression profiles are relatively stable overall, although gradual changes are observed for some cadherins. In the first part of the Results section, we will describe the ontogenetic expression of each cadherin in the primary visual cortex in detail. Figures 1–3 give an overview of the expression of each cadherin at representative stages of development. Figures 4 and 5 summarize the expression patterns for selected stages of development in schematic diagrams.

Figure 1.

Expression mapping in the layers of the primary visual cortex with cRNA probes for cadherin-4/R-cadherin (CDH4; A), cadherin-6 (CDH6; B), cadherin-7 (CDH7; C), cadherin-8 (CDH8; D), and cadherin-11 (CDH11; E). In situ hybridization was carried out at different embryonic stages (E) and postnatal stages (P). Developmental stages are indicated at the bottom of each panel. The layers are indicated at the right of each panel. To facilitate the identification of cortical layers, a thionin (Nissl) stain of an adjacent section is shown to the right of the in situ hybridization results for selected stages. I–VI, cortical layers I–VI; CP, cortical plate; IZ, intermediate zone; PP, preplate; SP, subplate; SPl, lower subplate; SPu, upper subplate; SVZ, subventricular zone; VZ, ventricular zone; WM, white matter. Scale bars are 25 μm for stage E23, 50 μm for stage E30, 100 μm for stage E38, 200 μm for stage P2, and 100 μm for stages from P13 to the adult stage.

Figure 1.

Expression mapping in the layers of the primary visual cortex with cRNA probes for cadherin-4/R-cadherin (CDH4; A), cadherin-6 (CDH6; B), cadherin-7 (CDH7; C), cadherin-8 (CDH8; D), and cadherin-11 (CDH11; E). In situ hybridization was carried out at different embryonic stages (E) and postnatal stages (P). Developmental stages are indicated at the bottom of each panel. The layers are indicated at the right of each panel. To facilitate the identification of cortical layers, a thionin (Nissl) stain of an adjacent section is shown to the right of the in situ hybridization results for selected stages. I–VI, cortical layers I–VI; CP, cortical plate; IZ, intermediate zone; PP, preplate; SP, subplate; SPl, lower subplate; SPu, upper subplate; SVZ, subventricular zone; VZ, ventricular zone; WM, white matter. Scale bars are 25 μm for stage E23, 50 μm for stage E30, 100 μm for stage E38, 200 μm for stage P2, and 100 μm for stages from P13 to the adult stage.

Figure 2.

Expression mapping in the layers of the primary visual cortex with cRNA probes for cadherin-14 (CDH14; A), cadherin-20 (CDH20; B), protocadherin-1 (PCDH1; C), protocadherin-7 (PCDH7; D), and protocadherin-8 (PCDH8; E). In situ hybridization was carried out at different embryonic stages (E) and postnatal stages (P). Developmental stages are indicated at the bottom of each panel. The layers are indicated at the right of each panel. To facilitate the identification of cortical layers, a thionin (Nissl) stain of an adjacent section is shown to the right of the in situ hybridization results for selected stages. I–VI, cortical layers I–VI; CP, cortical plate; IZ, intermediate zone; PP, preplate; SP, subplate; SPl, lower subplate; SPu, upper subplate; SVZ, subventricular zone; VZ, ventricular zone; WM, white matter. Scale bars are 25 μm for stage E23, 50 μm for stage E30, 100 μm for E38, 200 μm for stage P2, and 100 μm for stages from P13 to the adult stage.

Figure 2.

Expression mapping in the layers of the primary visual cortex with cRNA probes for cadherin-14 (CDH14; A), cadherin-20 (CDH20; B), protocadherin-1 (PCDH1; C), protocadherin-7 (PCDH7; D), and protocadherin-8 (PCDH8; E). In situ hybridization was carried out at different embryonic stages (E) and postnatal stages (P). Developmental stages are indicated at the bottom of each panel. The layers are indicated at the right of each panel. To facilitate the identification of cortical layers, a thionin (Nissl) stain of an adjacent section is shown to the right of the in situ hybridization results for selected stages. I–VI, cortical layers I–VI; CP, cortical plate; IZ, intermediate zone; PP, preplate; SP, subplate; SPl, lower subplate; SPu, upper subplate; SVZ, subventricular zone; VZ, ventricular zone; WM, white matter. Scale bars are 25 μm for stage E23, 50 μm for stage E30, 100 μm for E38, 200 μm for stage P2, and 100 μm for stages from P13 to the adult stage.

Figure 3.

Expression mapping in the layers of the primary visual cortex with cRNA probes for protocadherin-9 (PCDH9; A), protocadherin-10 (PCDH10; B), protocadherin-11 (PCDH11; C), protocadherin-17 (PCDH17; D), and protocadherin-19 (PCDH19; E). In situ hybridization was carried out at different embryonic stages (E) and postnatal stages (P). Developmental stages are indicated at the bottom of each panel. The layers are indicated at the right of each panel. To facilitate the identification of cortical layers, a thionin (Nissl) stain of an adjacent section is shown to the right of the in situ hybridization results for selected stages. I–VI, cortical layers I–VI; CP, cortical plate; IZ, intermediate zone; PP, preplate; SP, subplate; SPl, lower subplate; SPu, upper subplate; SVZ, subventricular zone; VZ, ventricular zone; WM, white matter. Scale bars are 25 μm for stage E23, 50 μm for stage E30, 100 μm for E38, 200 μm for stage P2, and 100 μm for stages from P13 to the adult stage.

Figure 3.

Expression mapping in the layers of the primary visual cortex with cRNA probes for protocadherin-9 (PCDH9; A), protocadherin-10 (PCDH10; B), protocadherin-11 (PCDH11; C), protocadherin-17 (PCDH17; D), and protocadherin-19 (PCDH19; E). In situ hybridization was carried out at different embryonic stages (E) and postnatal stages (P). Developmental stages are indicated at the bottom of each panel. The layers are indicated at the right of each panel. To facilitate the identification of cortical layers, a thionin (Nissl) stain of an adjacent section is shown to the right of the in situ hybridization results for selected stages. I–VI, cortical layers I–VI; CP, cortical plate; IZ, intermediate zone; PP, preplate; SP, subplate; SPl, lower subplate; SPu, upper subplate; SVZ, subventricular zone; VZ, ventricular zone; WM, white matter. Scale bars are 25 μm for stage E23, 50 μm for stage E30, 100 μm for E38, 200 μm for stage P2, and 100 μm for stages from P13 to the adult stage.

Figure 4.

Schematic diagram of the cadherin expression patterns in the layers of the primary visual cortex at embryonic day 23 (E23; A), E30 (B), E38 (C), and postnatal day 2 (P2; D). The layers are indicated at the left side of the panels. Layer-specific staining is represented by different colors. The intensity of the colors indicates the approximate general level of expression. CP, cortical plate; IZ, intermediate zone; PP, preplate; SP, subplate; SPl, lower subplate; SPu, upper subplate; SVZ, subventricular zone; VZ, ventricular zone.

Figure 4.

Schematic diagram of the cadherin expression patterns in the layers of the primary visual cortex at embryonic day 23 (E23; A), E30 (B), E38 (C), and postnatal day 2 (P2; D). The layers are indicated at the left side of the panels. Layer-specific staining is represented by different colors. The intensity of the colors indicates the approximate general level of expression. CP, cortical plate; IZ, intermediate zone; PP, preplate; SP, subplate; SPl, lower subplate; SPu, upper subplate; SVZ, subventricular zone; VZ, ventricular zone.

Figure 5.

Schematic diagram of the cadherin expression patterns in the layers of the primary visual cortex at postnatal day 13 (P13; A), P33 (B), and P60/adult (C). The layers are indicated at the left side of the panels. Layer-specific staining is represented by different colors. The intensity of the colors indicates the approximate general level of expression. The dotted patterns for CDH7 and PCDH8 indicates that a small subset of large cells is labeled. I–VI, cortical layers I–VI.

Figure 5.

Schematic diagram of the cadherin expression patterns in the layers of the primary visual cortex at postnatal day 13 (P13; A), P33 (B), and P60/adult (C). The layers are indicated at the left side of the panels. Layer-specific staining is represented by different colors. The intensity of the colors indicates the approximate general level of expression. The dotted patterns for CDH7 and PCDH8 indicates that a small subset of large cells is labeled. I–VI, cortical layers I–VI.

The expression pattern described is specific for the primary visual cortex of the ferret. Other cortical areas express the same cadherins, but in different layer-specific patterns (Krishna-K. and Christoph Redies, unpublished data). To demonstrate the specific expression profile in primary visual cortex, we examined the boundary between the primary and secondary visual cortex in the second part of the Results section (Figs 6, 7).

Figure 6.

Expression mapping of cadherins at the boundary between primary (V1) and secondary visual cortex (V2) at postnatal day 13 (P13; AE) and at P60 (FM). Adjacent sections were hybridized with cRNA probes for cadherin-8 (CDH8; B, I), cadherin-11 (CDH11; C), cadherin-20 (CDH20; D), protocadherin-10 (PCDH10; E), cadherin-4 (CDH4; G), cadherin-7 (CDH7; H), cadherin-14 (CDH14; J), protocadherin-7 (PCDH7; K), protocadherin-17 (PCDH17; L), and protocadherin-19 (PCDH19; M). The boundary between V1 and V2 is indicated by arrowheads. To facilitate the identification of the boundary, a Nissl stain (Thionin) of adjacent sections is shown in A and F, respectively. Scale bars are 300 μm (in A for AE) and 400 μm (in F for FM).

Figure 6.

Expression mapping of cadherins at the boundary between primary (V1) and secondary visual cortex (V2) at postnatal day 13 (P13; AE) and at P60 (FM). Adjacent sections were hybridized with cRNA probes for cadherin-8 (CDH8; B, I), cadherin-11 (CDH11; C), cadherin-20 (CDH20; D), protocadherin-10 (PCDH10; E), cadherin-4 (CDH4; G), cadherin-7 (CDH7; H), cadherin-14 (CDH14; J), protocadherin-7 (PCDH7; K), protocadherin-17 (PCDH17; L), and protocadherin-19 (PCDH19; M). The boundary between V1 and V2 is indicated by arrowheads. To facilitate the identification of the boundary, a Nissl stain (Thionin) of adjacent sections is shown in A and F, respectively. Scale bars are 300 μm (in A for AE) and 400 μm (in F for FM).

Figure 7.

Tyramide signal-amplified fluorescent in situ hybridization at the boundary between primary (V1) and secondary visual cortex (V2) at postnatal day 13 (P13). The section was doubly hybridized in situ with cRNA probes for cadherin-8 (CDH8; red) and protocadherin-10 (PCDH10; green). The boundary between V1 and V2 is indicated by arrowheads. The scale bar is 1 mm.

Figure 7.

Tyramide signal-amplified fluorescent in situ hybridization at the boundary between primary (V1) and secondary visual cortex (V2) at postnatal day 13 (P13). The section was doubly hybridized in situ with cRNA probes for cadherin-8 (CDH8; red) and protocadherin-10 (PCDH10; green). The boundary between V1 and V2 is indicated by arrowheads. The scale bar is 1 mm.

Cells in white matter and embryonic blood vessels were also found to express several of the cadherins studied. A detailed analysis of these cells is beyond the scope of the present work and will be published elsewhere (Krishna-K. and Christoph Redies, unpublished data).

Expression in Primary Visual Cortex (V1)

Cadherin-4 (CDH4)

Expression of CDH4 (R-cadherin) is already prominent and ubiquitous at the earliest stage examined (E23; Fig. 1A). Notably, the cells in the preplate express CDH4 strongly. From E30 to P2, strong expression persists in the ventricular zone. The subventricular zone, intermediate and MZs are moderately positive. The cortical plate is positive in superficial and deep layers. At P13, cells in the MZ (prospective layer I) and in layer II show prominent signal for CDH4 and layers IV–VI show moderate signal. From P33 to the adult stage, layer I cells, a subset of cells in layer II, and layers IV and VI remain positive.

Cadherin-6 (CDH6)

At E23, only the ventricular zone (neuroepithelium) expresses CDH6. At E30 and E38, expression in the entire cortical mantle is weak. At P2, moderate expression is observed in the cortical plate, subplate, and MZ (Fig. 1B). The cortical plate is more strongly positive in the superficial and deep laminae. At P13, CDH6-positive cells are scattered in all cortical layers, except in layer I, which remains negative until stage P46. During development of the cortical plate, expression becomes gradually restricted to particular layers. At P25, layers II–IV and VI contain numerous CDH6-positive cells, whereas positive cells are scarce in layer V. At P33, only a few cells remain positive in layer IV. Thereafter, the number of CDH6-positive cells in layers II and III decreases. At P60 and in the adult, expression is restricted to numerous cells in layer VI, to layer I cells and to very few, large cells in layer V.

Cadherin-7 (CDH7)

At the occipital pole of the cerebral cortex, weak staining is first observed at P2 in the upper layers of the cortical plate (Fig. 1C). A few scattered cells are also found in the other germinal layers. Staining then increases until it is moderately strong in layers II/III at P25. From P25 to the adult stage, signal in layers II/III decreases to low levels. In addition, from P13 to P60, a few scattered CDH7-positive cells with large somata are found in all cortical layers. In the adult, scattered CDH7-positive cells remain visible in all cortical layers. Layer I shows a weak staining only for stage P60 and in the adult.

Cadherin-8 (CDH8)

Moderate staining is observed first at E30 in the outer half of the cortical plate, ventricular zone and subventricular zone. At this and later stages, all other layers of V1 do not show signal (Fig. 1D). At E38 and P2, the signal in the cortical plate has become stronger. At P13, most cells in layers II–VI express CDH8; the most strongly labeled cells are located in layer IV. At P25, layer VI cells are still positive, but in layer V, only a few cells of large size retain signal. The expression profile observed at P25 persists until the adult stage.

Cadherin-11 (CDH11)

CDH11 is expressed by the cells in the preplate at E23. Apart from this, cells in the cortical plate, subplate, intermediate zone, subventricular zone and ventricular zone show weak to moderate expression of CDH11 from E30 onwards. At P2, the deeper layers of the cortical plate exhibit stronger staining than the upper layers (Fig. 1E). From P13 to P33, layers II/III and VI contain many CDH11-positive cells, whereas only few cells express CDH11 in layers IV and V. Expression becomes stronger as development proceeds. In particular, the number of positive cells in layer V increases from P46 to the adult stage. Overall, expression in infragranular layers is stronger than in supragranular layers. Also there is a moderate expression of CDH11 in the MZ and layer I from stage E30 to the adult stage.

Cadherin-14 (CDH14)

Initially, the preplate shows signal for CDH14 at E23. This signal persists in the MZ until E38. At P2, weak signal is seen in the upper subplate, intermediate zone and ventricular zone (Fig. 2A). Expression becomes more prominent at P25 when signal appears in layers II and VI; layers III and V contain a few positive cells. This expression profile persists till the adult stage. At stage P60 and in the adult, layer I also shows signal.

Cadherin-20 (CDH20)

Already at E23, the entire ventricular layer of the occipital pole shows strong signal. At E30, the other layers of the developing cortical mantle, including the subventricular zone, are negative (Fig. 2B). The signal in the ventricular zone has receded to the subependymal layer at E38 and P2. From P13 to P33, layers II/III and a few large pyramidal cells in layer V show CDH20 signal. Expression in layers II/III becomes weaker until the adult stage, especially in layer III, where only a subpopulation of cells is positive. Layer I cells are also positive from P2 to the adult stage. Strongly labeled cells persist in layer V until the adult stage.

Protocadherin-1 (PCDH1)

At E23, the ventricular zone is positive (Fig. 2C). At E38 and P2, the innermost (subependymal) lamina of the ventricular zone shows the strongest signal. Beginning at E38, the deeper layers of the cortical plate are PCDH1 positive. At P13, when the layering of the cortical plate becomes more distinct, cells in the supragranular layers (II/III) and the infragranular layers (V/VI) express PCDH1, although a few scattered cells are positive in layer IV as well. This pattern of expression remains basically the same from P13 onwards, but layers II and VI are more strongly stained in the adult. Layer I cells are moderately positive from P2 until the adult stage.

Protocadherin-7 (PCDH7)

Expression is first seen at E23 in the preplate. For other zones, expression starts at E30, when moderate signal is seen in the cortical plate, upper layers of the subventricular zone and intermediate zone (Fig. 2D). The signal in the cortical plate becomes considerably stronger during further development. Some cells are also positive in the upper subplate at P2. At P13, PCDH7 is expressed by cells in all layers of the cortical plate. From P25 to the adult stage, expression in layer IV becomes weak, whereas prominent signal persists in layers II, III, V, and VI. Cells in the MZ and in layer I are also positive from E30 to the adult stage.

Protocadherin-8 (PCDH8)

The cortical mantle is negative at E30. At P2, the ventricular, subventricular and intermediate zones, the subplate and the upper layers of the cortical plate are positive (Fig. 2E). From P13 to the adult, cells in layer II show prominent signal. In addition, from P2 to the adult stage, a few scattered positive cells are also found in all other layers. Layer I cells are also positive from P2 to the adult stage.

Protocadherin-9 (PCDH9)

Strong signal for PCDH9 is seen in the subventricular zone and in the preplate at E23 (Figs 3A and 4E). In addition, the cortical plate is also positive from its inception (E30). At E38, the ventricular and subventricular zones show prominent signal. The intermediate zone and lower subplate express PCDH9 weakly. Also, the cortical plate is strongly positive at E38 and at P2; prominent signal is seen in the deeper layers, including the upper subplate at P2. Signal in the ventricular layer has become weaker. From P25 to the adult stage, all cortical layers show expression; cells in layers III/IV and VI are the most strongly labeled. The MZ and layer I cells are also moderately positive from E30 to the adult stage.

Protocadherin-10 (PCDH10)

At E23, expression is seen in the preplate. For the other zones, staining is observed at E30 in the upper cortical plate, subplate and intermediate zone (Fig. 3B). Signal can be detected in the deep layers of the cortical plate, including the upper subplate, and the ventricular layer at E38 and P2. At P13, the newly formed layers II/III and IV are strongly PCDH10 positive. Staining in layer VI persists, whereas expression in layer V is weaker. From P13 to P60, this expression profile remains basically the same, but signal in layers II and III becomes less strong. At the adult stage, almost all cells in layers II–VI express PCDH10, except for some cells in layer V. Layer I cells are also positive from P25 to the adult stage (data not shown).

Protocadherin-11 (PCDH11)

At E30, a moderate staining is observed in the ventricular zone, the subventricular zone, subplate and the cortical plate (Fig. 3C). In addition, at E38, signal appears in the intermediate zone and the signal has intensified in the ventricular zone. From P13 to P46, expression in the developing cortical plate is most prominent in layers IV and VI. At P60 and in the adult, almost all cortical cells express PCDH11 homogeneously, except in layer I.

Protocadherin-17 (PCDH17)

At E23, the meninges show a strong signal for PCDH17. Expression in the cortical mantle begins at E30, when the subependymal layer of the ventricular zone shows strong signal; the subventricular zone and the cortical plate are only moderately positive (Fig. 3D). At E38 and P2, expression in the cortical plate is rather homogeneous and moderately strong and a few positive cells are seen in the intermediate zone. From P13 onwards, expression becomes restricted to cortical layers II/III and V/VI; later in development, expression is strongest in layers II and VI. This is the expression profile also seen in the adult.

Protocadherin-19 (PCDH19)

Expression sets in at E30, when weak signal is observed first in the cortical plate, subventricular zone and ventricular zone. Later in development, signal becomes stronger, especially in the deeper layers (Fig. 3E). Layers V/VI remain positive until stage P33. In addition, layer II is weakly labeled from P25 onwards. From P46 to the adult stage, there are strongly positive, large cells in layer V; cells in the other layers are only weakly or moderately stained.

Expression at the V1/V2 Boundary

A comparison of the cadherin expression patterns between V1 and the secondary visual cortex (V2) in a series of consecutive sections reveals that the layer-specific expression of most cadherins differs between the 2 areas, thereby allowing a demarcation of the V1/V2 boundary. Figures 6 and 7 show the V1/V2 boundary at P13 and at P60.

At P13, CDH8 expression is particularly strong in layers IV–VI of V2, but more moderate and ubiquitous in V1 (Fig. 6B). CDH11 is strongly expressed only in layers V/VI of V2; in V1, the same layer is weakly positive but, in addition, layers II/III express CDH11 (Fig. 6C). Unlike V1, V2 does not express CDH20 (Fig. 6D). Similarly, expression of PCDH10 falls off at the V1/V2 area (Fig. 6E). Double-labeling of a single section for both CDH8 and PCDH10 mRNA confirms the abrupt transition of the staining patterns at the boundary between V1 and V2 (Fig. 7).

A similar regional difference in cadherin expression can be observed at P60 (Fig. 6F–M). For example, CDH4 expression is prominent in layer V of V2 but absent from the same layer of V1 (Fig. 6G). CDH7 shows no significant expression for V2 in any of its layers, whereas strongly labeled cells are dispersed in all layers of V1 (Fig. 6H). CDH8 is generally expressed more strongly in V2 than in V1 (Fig. 6I). Layer IV is positive for CDH14 signal in V2 but not in V1 (Fig. 6J). PCDH7 shows weak staining in all layers of V2; in V1, expression is generally stronger but absent from layer IV (Fig. 6K). PCDH17 shows overall strong signal in all layers of V2; in contrast, there is weak expression in layers IV and V of V1, but strong expression only in layer VI (Fig. 6L). Strongly PCDH19-positive cells are absent in layer V of V2 but present in V1 (Fig. 6M).

Discussion

For the first time, 15 members of 2 cadherin subfamilies, classic cadherins and δ-protocadherins, were cloned and sequenced from the ferret brain, in order to study their expression in the developing primary visual cortex. Results from in situ hybridization revealed that the expression of the cadherins is subject to a tight temporal, layer-specific and region-specific regulation during corticogenesis. Not only the layers of the differentiating and mature cortical plate, but also the germinal zones of the early embryonic cortical mantle express the cadherins differentially. In addition, we provide evidence that some of the cadherins are expressed in subtypes of cells dispersed in specific cortical layers or throughout all cortical layers. The present identification of cadherins as a set of markers for cortical layers and neuronal subpopulations adds to the goal of obtaining a panel of markers that allows a comprehensive analysis of all neuron types in the mammalian neocortex (Hevner 2007).

Cadherins Provide a Code of Potentially Adhesive Cues for the Developing and Adult Ferret Primary Visual Cortex

Each of the 15 cadherins studied exhibits a unique, spatially restricted expression pattern that is dissimilar from that of other cadherins, although partial overlap between the cadherins is observed. The expression patterns are relatively stable throughout development. Changes in layer-specific expression are usually minor, if they occur at all, and take place slowly during development (Figs 1–3).

The cadherin-based code for specifying cortical laminae is probably a combinatorial one because each lamina is characterized by the expression of a subset of multiple cadherins (Figs 4, 5). Similar results have been obtained for other layered central nervous system structures, for example for the retina (Matsunaga et al. 1988; Wöhrn et al. 1998; Faulkner-Jones et al. 1999; Ruan et al. 2006) and the chicken tectum (Wöhrn et al. 1999).

Most of the cadherins studied here are expressed also in various other regions throughout the ferret brain and in neural circuits outside the visual system (Krishna-K. and Christoph Redies, unpublished data). A similarly widespread, but region-specific expression of cadherins was described before in the embryonic and postnatal brain of other vertebrates (Redies et al. 1993; Korematsu and Redies 1997; Suzuki et al. 1997; Hirano et al. 1999; Redies et al. 2000, 2005; Obst-Pernberg et al. 2001; Bekirov et al. 2002; Vanhalst et al. 2005; Kim et al. 2007). Based on these results, it has been proposed that cadherins provide an adhesive code for developing brain structures, neural circuits and synapses (for reviews, see Redies 1997, 2000; Hirano et al. 2003; Takeichi 2007).

Layer-Specific Expression of Cadherins in the Visual Cortex

As summarized in Figure 4, cadherins are markers for the different embryonic germinal zones of the developing cortical mantle. For example, the cells of the E23 preplate express CDH4, CDH11, CDH14, PCDH7, PCDH9, and PCDH10. Possibly, at least some of the cadherin-expressing cells in the preplate (prospective layer I) are Cajal-Retzius. The subependymal layer that is marked by the combinatorial expression of CDH7, CDH14, PCDH17, and PCDH19, and the ventricular zone expresses CDH4, CDH11, CDH20, PCDH1, and PCDH11. Previously, 2 classic cadherins, N-cadherin and CDH6, have been shown to play roles in the maintenance of the neuroepithelial layer, the proper lamination of neural tissue and the migration of neural cells (Barami et al. 1994; Radice et al. 1997; Gänzler-Odenthal and Redies 1998; Coles et al. 2007; Ruan et al. 2006; Kadowaki et al. 2007). Moreover, the cadherin-mediated adhesive system regulates cell proliferation and cell death in the neuroepithelium (Babb et al. 2005; Lien et al. 2006; Noles and Chenn 2007). It remains unclear at present whether the cadherins investigated in this study have similar functions.

A large number of other genes like Emx1, Emx2, Pax6, Dlx-2, IgCAM, and MDGA1 are expressed in a layer-specific fashion in the germinal zones (Bulfone et al. 1993; Panganiban and Rubenstein 2002; Bishop et al. 2003; Hevner et al. 2003; Takeuchi et al. 2007) and in the differentiating and mature cortical plate (for a review, see Funatsu et al. 2004). The cadherins studied in the present work are the first molecules from a single gene family that differentially distinguish the various germinal zones during development. In addition, most of the previously described genes code for gene regulatory proteins and are involved in embryonic pattern formation. In contrast, cadherins are a family of morphoregulatory molecules, possibly acting downstream of genetic patterning mechanisms (Shimamura et al. 1994; Stoykova et al. 1997; Miyashita-Lin et al. 1999; Nakagawa et al. 1999; Rubenstein et al. 1999; Bishop et al. 2000; Bishop et al. 2002; Garel et al. 2003; Luo et al. 2006; Rasin et al. 2007).

Cadherins are likely to be involved also in target recognition and intracortical circuit formation during corticogenesis. It has been shown previously that pre- and postsynaptic neurons often express the same cadherin in a matching fashion (Redies et al. 1993; Wöhrn et al. 1998; for a reviews, see Redies 1997, 2000; Hirano et al. 2003). For example, thalamic afferents and their cortical targets were shown to express matching cadherins in the cerebral cortex of rodents (Suzuki et al. 1997; Gil et al. 2002; Kim et al. 2007). In the chicken optic tectum, N-cadherin mediates layer-specific target recognition (Yamagata et al. 1995). It is possible, but remains to be demonstrated experimentally, that the expression of cadherins observed in the present study also regulates the formation of layer-specific cortical connectivity. The persistence of cadherin expression in the adult visual cortex supports the notion that cadherins play a role also in mature cortical function. Several cadherins have been found at the synapse, also during synaptogenesis (Fannon and Colman 1996; Uchida et al. 1996; Bozdagi et al. 2000; Togashi et al. 2002). A role for cadherins in dendritic sprouting and synapse plasticity has been proposed (for a review, see Takeichi 2007). We are currently generating antibodies against some of the cadherins in order to localize the cadherin proteins at the synapse also in the ferret visual cortex.

The function of classic cadherins and δ-protocadherins in the above processes may be different, as suggested by differences in the intracellular binding partners. Classic cadherins are linked intracellularly to a variety of molecules, including some catenins, which play a role in signal transduction and gene regulation; for example, one of the binding partners, β-catenin, is an integral part of the canonical Wnt pathway (for reviews, see Hirano et al. 2003; Nelson and Nusse 2004). Known intracellular binding partners of δ-protocadherins include the synaptic molecule protein phosphatase 1α, TAF1/set, β-catenin, Xfz7 and mDab1 (for a review, see Redies et al. 2005).

Region-Specific Expression of Cadherins in Primary Visual Cortex

Previous studies demonstrated that cadherins are regional markers for cortical areas during early embryonic development of the mouse. Examples are N-cadherin, Cdh4, Cdh6, Cdh8, and Cdh11 and several δ-protocadherins (Korematsu and Redies 1997; Suzuki et al. 1997; Simonneau and Thiery 1998; Rubenstein et al. 1999; Obst-Pernberg et al. 2001; Bekirov et al. 2002; Kim et al. 2007). In the present study, we show that differences in cadherin expression demarcate the boundary of the V1 and V2 subregions within the visual cortex. Cadherin expression thus reflects the functional compartmentation of the cerebral cortex into functional subregions. The V1/V2 boundary is also marked by the expression of other molecules like Cat-301, alkaline phosphatase and cytochrome oxidase (Hockfield et al. 1990; Fonta and Imbert 2002).

One particularly striking feature of ferret visual cortex is its columnar functional architecture (Redies et al. 1990; Chapman et al. 1996; Weliky et al. 1996). Surprisingly, at this highest level of cortical regionalization, we did not obtain any evidence for a differential expression of cadherins at the mRNA level.

Cadherin Expression by Subsets of Cortical Neurons

A closer look at the expression of cadherins reveals that not all cadherins are expressed by all neurons in a given cortical layer. A particularly striking example is the expression of CDH7 by scattered cells in all cortical layers (Figs 1C, 5, and 6). It is conceivable that these cells represent a particular type of neurons, for example interneurons. This suggestion, which requires confirmation by a double-labeling study with interneurons markers, is supported by the finding that similarly distributed cells are found in other cortical areas.

In some cortical layers, subsets of neurons express a particular cadherin. For example, CDH4, CDH6, CDH7, CDH8, CDH11, CDH14, CDH20, PCDH1, PCDH8, PCDH9, and PCDH19 mark subsets of neurons in layer V. This layer contains a mixture of pyramidal neurons, which project to various subcortical targets. It has been shown previously in the chicken tectum that subpopulations of projection neurons and their axons express cadherins differentially (Wöhrn et al. 1999) and that the cadherins target the tectofugal axons to specific axonal pathways (Treubert-Zimmermann et al. 2004). Future studies employing antibodies will show whether a similar differential cadherin expression is also implemented in the subcortical fiber projection systems that originate in the ferret visual cortex. Another possibility is that cadherins, which are expressed by subsets of neurons in a given cortical layer, mediate the formation of intracortical microcircuitry, as has been proposed for α-protocadherins (Kohmura et al. 1998).

Funding

Interdisciplinary Clinical Research Center of the University of Jena (IZKF Jena, TP 1-16); and the German Research Council (DFG Re 616/4-4).

Ferrets were kindly provided by Dr Dieter Wolff and his colleagues at the Federal Institute of Risk Research in Berlin-Marienfelde, Germany. We thank Dr Jiankai Luo and Ms Heike Thieme for their help in sequencing, Ms Jessica Heyder and Ms Sylvia Hänßgen for expert technical assistance, Dr Jayachandran Gopalakrishnan for help in the initial part of the study, Dr Marcus Frank for helpful suggestions for designing the degenerate primers, and members of the laboratory for discussion. Conflict of Interest: None declared.

References

Anderson
SA
Eisenstat
DD
Shi
L
Rubenstein
JL
Interneuron migration from basal forebrain to neocortex: dependence on Dlx genes
Science.
 , 
1997
, vol. 
278
 (pg. 
474
-
476
)
Angevine
JB
Jr
Sidman
RL
Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse
Nature.
 , 
1961
, vol. 
192
 (pg. 
766
-
768
)
Babb
SG
Kotradi
SM
Shah
B
Chiappini-Williamson
C
Bell
LN
Schmeiser
G
Chen
E
Liu
Q
Marrs
JA
Zebrafish R-cadherin (Cdh4) controls visual system development and differentiation
Dev Dyn.
 , 
2005
, vol. 
233
 (pg. 
930
-
945
)
Barami
K
Kirschenbaum
B
Lemmon
V
Goldman
SA
N-cadherin and Ng-CAM/8D9 are involved serially in the migration of newly generated neurons into the adult songbird brain
Neuron.
 , 
1994
, vol. 
13
 (pg. 
567
-
582
)
Bekirov
IH
Needleman
LA
Zhang
W
Benson
DL
Identification and localization of multiple classic cadherins in developing rat limbic system
Neuroscience.
 , 
2002
, vol. 
115
 (pg. 
213
-
227
)
Bishop
KM
Goudreau
G
O'Leary
DD
Regulation of area identity in the mammalian neocortex by Emx2 and Pax6
Science.
 , 
2000
, vol. 
288
 (pg. 
344
-
349
)
Bishop
KM
Garel
S
Nakagawa
Y
Rubenstein
JL
O'Leary
DD
Emx1 and Emx2 cooperate to regulate cortical size, lamination, neuronal differentiation, development of cortical efferents, and thalamocortical pathfinding
J Comp Neurol.
 , 
2003
, vol. 
457
 (pg. 
345
-
360
)
Bishop
KM
Rubenstein
JL
O'Leary
DD
Distinct actions of Emx1, Emx2, and Pax6 in regulating the specification of areas in the developing neocortex
J Neurosci.
 , 
2002
, vol. 
22
 (pg. 
7627
-
7638
)
Bozdagi
O
Shan
W
Tanaka
H
Benson
DL
Huntley
GW
Increasing numbers of synaptic puncta during late-phase LTP: N-cadherin is synthesized, recruited to synaptic sites, and required for potentiation
Neuron.
 , 
2000
, vol. 
28
 (pg. 
245
-
259
)
Bulfone
A
Kim
HJ
Puelles
L
Porteus
MH
Grippo
JF
Rubenstein
JL
The mouse Dlx-2 (Tes-1) gene is expressed in spatially restricted domains of the forebrain, face and limbs in midgestation mouse embryos
Mech Dev.
 , 
1993
, vol. 
40
 (pg. 
129
-
140
)
Caviness
VS
Jr
Takahashi
T
Nowakowski
RS
Numbers, time and neocortical neuronogenesis: a general developmental and evolutionary model
Trends Neurosci.
 , 
1995
, vol. 
9
 (pg. 
379
-
383
)
Chapman
B
Stryker
MP
Bonhoeffer
T
Development of orientation preference maps in ferret primary visual cortex
J Neurosci.
 , 
1996
, vol. 
16
 (pg. 
6443
-
6453
)
Coles
EG
Taneyhill
LA
Bronner-Fraser
M
A critical role for cadherin 6B in regulating avian neural crest emigration
Dev Biol.
 , 
2007
, vol. 
312
 (pg. 
533
-
544
)
Fannon
AM
Colman
DR
A model for central synaptic junctional complex formation based on the differential adhesive specificities of the cadherins
Neuron.
 , 
1996
, vol. 
17
 (pg. 
423
-
434
)
Faulkner-Jones
BE
Godinho
LN
Reese
BE
Pasquini
GF
Ruefli
A
Tan
SS
Cloning and expression of mouse cadherin-7, a type-II cadherin isolated from the developing eye
Mol Cell Neurosci.
 , 
1999
, vol. 
14
 (pg. 
1
-
16
)
Fonta
C
Imbert
M
Vascularization in the primate visual cortex during development
Cereb Cortex.
 , 
2002
, vol. 
12
 (pg. 
199
-
211
)
Frank
M
Kemler
R
Protocadherins
Curr Opin Cell Biol.
 , 
2002
, vol. 
14
 (pg. 
557
-
562
)
Funatsu
N
Inoue
T
Nakamura
S
Gene expression analysis of the late embryonic mouse cerebral cortex using DNA microarray: identification of several region- and layer-specific genes
Cereb Cortex.
 , 
2004
, vol. 
14
 (pg. 
1031
-
1044
)
Gaitan
Y
Bouchard
M
Expression of the delta-protocadherin gene Pcdh19 in the developing mouse embryo
Gene Expr Patterns.
 , 
2006
, vol. 
6
 (pg. 
893
-
899
)
Gänzler-Odenthal
SI
Redies
C
Blocking N-cadherin function disrupts the epithelial structure of differentiating neural tissue in the embryonic chicken brain
J Neurosci.
 , 
1998
, vol. 
18
 (pg. 
5415
-
5425
)
Garel
S
Huffman
KJ
Rubenstein
JL
Molecular regionalization of the neocortex is disrupted in Fgf8 hypomorphic mutants
Development.
 , 
2003
, vol. 
130
 (pg. 
1903
-
1914
)
Gil
OD
Needleman
L
Huntley
GW
Developmental patterns of cadherin expression and localization in relation to compartmentalized thalamocortical terminations in rat barrel cortex
J Comp Neurol.
 , 
2002
, vol. 
453
 (pg. 
372
-
388
)
Hertel
N
Krishna-
K
Nuernberger
M
Redies
C
A cadherin-based code for the divisions of the mouse basal ganglia
J Comp Neurol.
 , 
2008
, vol. 
508
 (pg. 
511
-
528
)
Hevner
RF
Layer-specific markers as probes for neuron type identity in human neocortex and malformations of cortical development
J Neuropathol Exp Neurol.
 , 
2007
, vol. 
66
 (pg. 
101
-
109
)
Hevner
RF
Daza
RA
Rubenstein
JL
Stunnenberg
H
Olavarria
JF
Englund
C
Beyond laminar fate: toward a molecular classification of cortical projection/pyramidal neurons
Dev Neurosci.
 , 
2003
, vol. 
25
 (pg. 
139
-
151
)
Hirano
S
Suzuki
ST
Redies
C
The cadherin superfamily in neural development: diversity, function and interaction with other molecules
Front Biosci.
 , 
2003
, vol. 
8
 (pg. 
306
-
355
)
Hirano
S
Yan
Q
Suzuki
ST
Expression of a novel protocadherin, OL-protocadherin, in a subset of functional systems of the developing mouse brain
J Neurosci.
 , 
1999
, vol. 
19
 (pg. 
995
-
1005
)
Hockfield
S
Tootell
RB
Zaremba
S
Molecular differences among neurons reveal an organization of human visual cortex
Proc Natl Acad Sci USA.
 , 
1990
, vol. 
87
 (pg. 
3027
-
3031
)
Jackson
CA
Peduzzi
JD
Hickey
TL
Visual cortex development in the ferret. I. Genesis and migration of visual cortical neurons
J Neurosci.
 , 
1989
, vol. 
9
 (pg. 
1242
-
1253
)
Kadowaki
M
Nakamura
S
Machon
O
Krauss
S
Radice
GL
Takeichi
M
N-cadherin mediates cortical organization in the mouse brain
Dev Biol.
 , 
2007
, vol. 
304
 (pg. 
22
-
33
)
Kim
SY
Chung
HS
Sun
W
Kim
H
Spatiotemporal expression pattern of non-clustered protocadherin family members in the developing rat brain
Neuroscience.
 , 
2007
, vol. 
147
 (pg. 
996
-
1021
)
Kohmura
N
Senzaki
K
Hamada
S
Kai
N
Yasuda
R
Watanabe
M
Ishii
H
Yasuda
M
Mishina
M
Yagi
T
Diversity revealed by a novel family of cadherins expressed in neurons at a synaptic complex
Neuron.
 , 
1998
, vol. 
20
 (pg. 
1137
-
1151
)
Korematsu
K
Redies
C
Restricted expression of cadherin-8 in segmental and functional subdivisions of the embryonic mouse brain
Dev Dyn.
 , 
1997
, vol. 
208
 (pg. 
178
-
189
)
Lien
WH
Klezovitch
O
Fernandez
TE
Delrow
J
Vasioukhin
V
alphaE-catenin controls cerebral cortical size by regulating the hedgehog signaling pathway
Science.
 , 
2006
, vol. 
311
 (pg. 
1560
-
1562
)
Luo
J
Ju
MJ
Redies
C
Regionalized cadherin-7 expression by radial glia is regulated by Shh and Pax7 during chicken spinal cord development
Neuroscience.
 , 
2006
, vol. 
142
 (pg. 
1133
-
1143
)
Matsunaga
M
Hatta
K
Takeichi
M
Role of N-cadherin cell adhesion molecules in the histogenesis of neural retina
Neuron.
 , 
1988
, vol. 
1
 (pg. 
289
-
295
)
McSherry
GM
Mapping of cortical histogenesis in the ferret
J Embryol Exp Morphol.
 , 
1984
, vol. 
81
 (pg. 
239
-
252
)
Miyashita-Lin
EM
Hevner
R
Wassarman
KM
Martinez
S
Rubenstein
JL
Early neocortical regionalization in the absence of thalamic innervation
Science.
 , 
1999
, vol. 
285
 (pg. 
906
-
909
)
Molyneaux
BJ
Arlotta
P
Menezes
JR
Macklis
JD
Neuronal subtype specification in the cerebral cortex
Nat Rev Neurosci.
 , 
2007
, vol. 
8
 (pg. 
427
-
437
)
Nadarajah
B
Parnavelas
JG
Modes of neuronal migration in the developing cerebral cortex
Nat Rev Neurosci.
 , 
2002
, vol. 
3
 (pg. 
423
-
432
)
Nakagawa
Y
Johnson
JE
O'Leary
DD
Graded and areal expression patterns of regulatory genes and cadherins in embryonic neocortex independent of thalamocortical input
J Neurosci.
 , 
1999
, vol. 
19
 (pg. 
10877
-
10885
)
Nelson
WJ
Nusse
R
Convergence of Wnt, beta-catenin, and cadherin pathways
Science.
 , 
2004
, vol. 
303
 (pg. 
1483
-
1487
)
Noles
SR
Chenn
A
Cadherin inhibition of beta-catenin signaling regulates the proliferation and differentiation of neural precursor cells
Mol Cell Neurosci.
 , 
2007
, vol. 
35
 (pg. 
549
-
558
)
Nollet
F
Kools
P
van Roy
F
Phylogenetic analysis of the cadherin superfamily allows identification of six major subfamilies besides several solitary members
J Mol Biol.
 , 
2000
, vol. 
299
 (pg. 
551
-
572
)
Obst-Pernberg
K
Medina
L
Redies
C
Expression of R-cadherin and N-cadherin by cell groups and fiber tracts in the developing mouse forebrain: relation to the formation of functional circuits
Neuroscience.
 , 
2001
, vol. 
106
 (pg. 
505
-
533
)
Panganiban
G
Rubenstein
JL
Developmental functions of the Distal-less/Dlx homeobox genes
Development.
 , 
2002
, vol. 
129
 (pg. 
4371
-
4386
)
Price
SR
De Marco Garcia
NV
Ranscht
B
Jessell
TM
Regulation of motor neuron pool sorting by differential expression of type II cadherins
Cell.
 , 
2002
, vol. 
109
 (pg. 
205
-
216
)
Radice
GL
Rayburn
H
Matsunami
H
Knudsen
KA
Takeichi
M
Hynes
RO
Developmental defects in mouse embryos lacking N-cadherin
Dev Biol.
 , 
1997
, vol. 
181
 (pg. 
64
-
78
)
Rakic
P
Neurons in rhesus monkey visual cortex: systematic relation between time of origin and eventual disposition
Science.
 , 
1974
, vol. 
183
 (pg. 
425
-
427
)
Rakic
P
Specification of cerebral cortical areas
Science.
 , 
1988
, vol. 
241
 (pg. 
170
-
176
)
Rakic
P
Caviness
VS
Jr
Cortical development: view from neurological mutants two decades later
Neuron.
 , 
1995
, vol. 
14
 (pg. 
1101
-
1104
)
Rash
BG
Grove
EA
Area and layer patterning in the developing cerebral cortex
Curr Opin Neurobiol.
 , 
2006
, vol. 
16
 (pg. 
25
-
34
)
Rasin
MR
Gazula
VR
Breunig
JJ
Kwan
KY
Johnson
MB
Liu-Chen
S
Li
HS
Jan
LY
Jan
YN
Rakic
P
, et al.  . 
Numb and Numbl are required for maintenance of cadherin-based adhesion and polarity of neural progenitors
Nat Neurosci
 , 
2007
, vol. 
10
 (pg. 
819
-
827
)
Redies
C
Cadherins and the formation of neural circuitry in the vertebrate CNS
Cell Tissue Res.
 , 
1997
, vol. 
290
 (pg. 
405
-
413
)-)
Redies
C
Cadherins in the central nervous system
Prog Neurobiol.
 , 
2000
, vol. 
61
 (pg. 
611
-
648
)
Redies
C
Ast
M
Nakagawa
S
Takeichi
M
Martínez-de-la-Torre
M
Puelles
L
Morphologic fate of diencephalic prosomeres and their subdivisions revealed by mapping cadherin expression
J Comp Neurol.
 , 
2000
, vol. 
421
 (pg. 
481
-
514
)
Redies
C
Diksic
M
Riml
H
Functional organization in the ferret visual cortex: a double-label 2-deoxyglucose study
J Neurosci.
 , 
1990
, vol. 
10
 (pg. 
2791
-
2803
)
Redies
C
Engelhart
K
Takeichi
M
Differential expression of N- and R-cadherin in functional neuronal systems and other structures of the developing chicken brain
J Comp Neurol.
 , 
1993
, vol. 
333
 (pg. 
398
-
416
)
Redies
C
Takeichi
M
Expression of N-cadherin mRNA during development of the mouse brain
Dev Dyn.
 , 
1993
, vol. 
197
 (pg. 
26
-
39
)
Redies
C
Takeichi
M
Cadherins in the developing central nervous system: an adhesive code for segmental and functional subdivisions
Dev Biol.
 , 
1996
, vol. 
180
 (pg. 
413
-
423
)
Redies
C
Vanhalst
K
Roy
F
delta-Protocadherins: unique structures and functions
Cell Mol Life Sci.
 , 
2005
, vol. 
62
 (pg. 
2840
-
2852
)
Rockland
KS
Anatomical organization of primary visual cortex (area 17) in the ferret
J Comp Neurol.
 , 
1985
, vol. 
241
 (pg. 
225
-
236
)
Ruan
G
Wedlich
D
Koehler
A
Xenopus cadherin-6 regulates growth and epithelial development of the retina
Mech Dev.
 , 
2006
, vol. 
123
 (pg. 
881
-
892
)
Rubenstein
JL
Anderson
S
Shi
L
Miyashita-Lin
E
Bulfone
A
Hevner
R
Genetic control of cortical regionalization and connectivity
Cereb Cortex.
 , 
1999
, vol. 
9
 (pg. 
524
-
532
)
Shimamura
K
Takahashi
T
Takeichi
M
Wnt-1-dependent regulation of local E-cadherin and alpha N-catenin expression in the embryonic mouse brain
Dev Biol.
 , 
1994
, vol. 
120
 (pg. 
2225
-
2234
)
Simonneau
L
Thiery
JP
The mesenchymal cadherin-11 is expressed in restricted sites during the ontogeny of the rat brain in modes suggesting novel functions
Cell Adhes Commun.
 , 
1998
, vol. 
6
 (pg. 
431
-
450
)
Stoykova
A
Götz
M
Gruss
P
Price
J
Pax6-dependent regulation of adhesive patterning, R-cadherin expression and boundary formation in developing forebrain
Development.
 , 
1997
, vol. 
124
 (pg. 
3765
-
3777
)
Suzuki
SC
Inoue
T
Kimura
Y
Tanaka
T
Takeichi
M
Neuronal circuits are subdivided by differential expression of type-II classic cadherins in postnatal mouse brains
Mol Cell Neurosci.
 , 
1997
, vol. 
9
 (pg. 
433
-
447
)
Takeichi
M
The cadherin superfamily in neuronal connections and interactions
Nat Rev Neurosci.
 , 
2007
, vol. 
8
 (pg. 
11
-
20
)
Takeuchi
A
Hamasaki
T
Litwack
ED
O'Leary
DD
Novel IgCAM, MDGA1, expressed in unique cortical area- and layer-specific patterns and transiently by distinct forebrain populations of Cajal-Retzius neurons
Cereb Cortex.
 , 
2007
, vol. 
17
 (pg. 
1531
-
1541
)
Togashi
H
Abe
K
Mizoguchi
A
Takaoka
K
Chisaka
O
Takeichi
M
Cadherin regulates dendritic spine morphogenesis
Neuron.
 , 
2002
, vol. 
35
 (pg. 
1
-
3
)
Treubert-Zimmermann
U
Heyers
D
Redies
C
Targeting axons to specific fiber tracts in vivo by altering cadherin expression
J Neurosci.
 , 
2002
, vol. 
22
 (pg. 
7617
-
7626
)
Uchida
N
Honjo
Y
Johnson
KR
Wheelock
MJ
Takeichi
M
The catenin/cadherin adhesion system is localized in synaptic junctions bordering transmitter release zones
J Cell Biol.
 , 
1996
, vol. 
135
 (pg. 
767
-
779
)
Vanhalst
K
Kools
P
Staes
K
van Roy
F
Redies
C
delta-Protocadherins: a gene family expressed differentially in the mouse brain
Cell Mol Life Sci.
 , 
2005
, vol. 
62
 (pg. 
1247
-
1259
)
Watakabe
A
Ichinohe
N
Ohsawa
S
Hashikawa
H
Komatsu
H
Rockland
KS
Yamamori
T
Comparative analysis of layer-specific genes in mammalian neocortex
Cereb Cortex.
 , 
2007
, vol. 
17
 (pg. 
1918
-
1933
)
Weliky
M
Bosking
WH
Fitzpatrick
D
A systematic map of direction preference in primary visual cortex
Nature.
 , 
1996
, vol. 
379
 (pg. 
725
-
728
)
Wöhrn
JC
Nakagawa
S
Ast
M
Takeichi
M
Redies
C
Combinatorial expression of cadherins in the tectum and the sorting of neurites in the tectofugal pathways of the chicken embryo
Neuroscience.
 , 
1999
, vol. 
90
 (pg. 
985
-
1000
)
Wöhrn
JC
Puelles
L
Nakagawa
S
Takeichi
M
Redies
C
Cadherin expression in the retina and retinofugal pathways of the chicken embryo
J Comp Neurol.
 , 
1998
, vol. 
396
 (pg. 
20
-
38
)
Yamagata
M
Herman
JP
Sanes
JR
Lamina-specific expression of adhesion molecules in developing chick optic tectum
J Neurosci.
 , 
1995
, vol. 
15
 (pg. 
4556
-
4571
)
Zhou
L
Jossin
Y
Goffinet
AM
Identification of small molecules that interfere with radial neuronal migration and early cortical plate development
Cereb Cortex.
 , 
2007
, vol. 
17
 (pg. 
211
-
220
)
Zou
C
Huang
W
Ying
G
Wu
Q
Sequence analysis and expression mapping of the rat clustered protocadherin gene repertoires
Neuroscience.
 , 
2007
, vol. 
144
 (pg. 
579
-
603
)