Cerebral Cortex Advance Access originally published online on June 4, 2008
Cerebral Cortex 2009 19(2):388-401; doi:10.1093/cercor/bhn090
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Layer-Specific Expression of Multiple Cadherins in the Developing Visual Cortex (V1) of the Ferret
1 Institute of Anatomy I, 2 Junior Research Group of Neurogenetics, University of Jena School of Medicine, Teichgraben 7, D-07743 Jena, Germany
Address correspondence to Christoph Redies MD, PhD, Institute of Anatomy I, University of Jena School of Medicine, Teichgraben 7, D-07743 Jena, Germany. Email: redies{at}mti.uni-jena.de.
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Abstract |
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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
-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.
Key Words: cell adhesion • cortical plate • corticogenesis • germinal zones • -protocadherins
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingReferences |
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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 
-, β-, and 
-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 -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), -protocadherins (Kohmura et al. 1998; Zou et al. 2007), -protocadherins (Zou et al. 2007), and -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 -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).
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Materials and Methods |
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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 -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.
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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], 3x saline-sodium citrate (SSC), 1x Denhardt's solution, 10x 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, 3x SSC, 1x Denhardt's solution, 10x 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).
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Results |
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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
-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.
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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).
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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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingReferences |
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For the first time, 15 members of 2 cadherin subfamilies, classic cadherins and
-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 -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 -protocadherins include the synaptic molecule protein phosphatase 1, 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 -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 -protocadherins (Kohmura et al. 1998).
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingReferences |
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Interdisciplinary Clinical Research Center of the University of Jena (IZKF Jena, TP 1-16); and the German Research Council (DFG Re 616/4-4).
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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.
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