Wilms' tumour (WT) is a pediatric tumor of the kidney that arises via failure of the fetal developmental program. The absence of identifiable mutations in the majority of WTs suggests the frequent involvement of epigenetic aberrations in WT. We therefore conducted a genome-wide analysis of promoter hypermethylation in WTs and identified hypermethylation at chromosome 5q31 spanning 800 kilobases (kb) and more than 50 genes. The methylated genes all belong to alpha-, beta-, and gamma-protocadherin (PCDH) gene clusters (Human Genome Organization nomenclature PCDHA@, PCDHB@, and PCDHG@, respectively). This demonstrates that long-range epigenetic silencing (LRES) occurs in developmental tumors as well as in adult tumors. Bisulfite polymerase chain reaction analysis showed that PCDH hypermethylation is a frequent event found in all Wilms' tumor subtypes. Hypermethylation is concordant with reduced PCDH expression in tumors. WT precursor lesions showed no PCDH hypermethylation, suggesting that de novo PCDH hypermethylation occurs during malignant progression. Discrete boundaries of the PCDH domain are delimited by abrupt changes in histone modifications; unmethylated genes flanking the LRES are associated with permissive marks which are absent from methylated genes within the domain. Silenced genes are marked with non-permissive histone 3 lysine 9 dimethylation. Expression analysis of embryonic murine kidney and differentiating rat metanephric mesenchymal cells demonstrates that Pcdh expression is developmentally regulated and that Pcdhg@ genes are expressed in blastemal cells. Importantly, we show that PCDHs negatively regulate canonical Wnt signalling, as short-interfering RNA-induced reduction of PCDHG@ encoded proteins leads to elevated beta-catenin protein, increased beta-catenin/T-cell factor (TCF) reporter activity, and induction of Wnt target genes. Conversely, over-expression of PCDHs suppresses beta-catenin/TCF-reporter activity and also inhibits colony formation and growth of cancer cells in soft agar. Thus PCDHs are candidate tumor suppressors that modulate regulatory pathways critical in development and disease, such as canonical Wnt signaling.

Protocadherins are cadherin-like molecules with adhesive and signaling functions, in particular, during neuronal development. Large protocadherin (Pcdh) gene clusters are present in the genome of vertebrates. In the zebrafish, two Pcdh clusters are found on chromosomes 10 (DrPcdh1) and 14 (DrPcdh2), each divided into subclusters of DrPcdhα and DrPcdhγ family genes. In total, about 100 different DrPcdh molecules are predicted. We have analyzed the expression of the four DrPcdh subclusters and find that DrPcdh transcripts are upregulated in the developing zebrafish nervous system. In the adult fish brain, all four DrPcdh clusters are expressed in differentiated neurons, in particular, in the thalamic nuclei, tectum, and cerebellum. We show that expression patterns grossly overlap for each cluster but with regional differences and variations in strength of expression. Strikingly, the DrPcdh2γ cluster, distinct from the three other clusters, is also expressed in neuronal precursor cells and ependymal cells of the embryonic and adult nervous system, as well as in specific non-neuronal epithelia. Antibodies to a conserved motif in the constant region of DrPcdh2γ stain fiber tracts and neuropil of the zebrafish brain and cell–cell junctions in epithelia. Our results indicate that multiple DrPcdhs of the different clusters are expressed in differentiated zebrafish neurons, suggesting evolutionarily conserved functions of protocadherin clusters in cell adhesion and signaling. In addition, DrPcdh2γ may exert more specific roles in neuronal precursor and non-neural epithelial cells, which have not yet been described for mammalian Pcdhγ. Thus, our findings in zebrafish open new perspectives to examine these functions in other vertebrate model organisms.

gamma-Protocadherins (Pcdh gamma) are type I transmembrane proteins, which are most notably expressed in the nervous system. They are enriched at synapses and involved in synapse formation, specification, and maintenance. In this study, we show that Pcdh gamma C3 and Pcdh gamma B4 are specifically cleaved within their ectodomains by the disintegrin and metalloprotease ADAM10. Analysis of ADAM10-deficient fibroblasts and embryos, inhibitor studies, as well as RNA interference-mediated down-regulation demonstrated that ADAM10 is not only responsible for the constitutive but also for the regulated shedding of these proteins in fibroblasts and in neuronal cells. In contrast to N-cadherin shedding, which was activated by N-methyl-D-aspartic acid receptor activation in neuronal cells, Pcdh gamma shedding was induced by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrate stimulation, suggesting differential regulation mechanisms of cadherin-mediated functions at synapses. Cell aggregation assays in the presence or absence of metalloprotease inhibitors strongly suggest that the ectodomain shedding events modulate the cell adhesion role of Pcdh gamma. The identification of ADAM10 as the protease responsible for constitutive and regulated Pcdh gamma shedding may therefore provide new insight into the regulation of Pcdh gamma functions.

Three tandemly arrayed protocadherin gene clusters (Pcdh-alpha, -beta -gamma) comprising more than 50 genes are found in human and mouse. Here, we have investigated the expression and distribution of individual gamma-protocadherins (Pcdhs-gamma) in the developing mouse brain. We find that transfection of Pcdh-gamma genes promotes calcium-dependent cell adhesion in HEK 293 cells. Furthermore, Pcdh-gamma can be recruited to synapses of transfected primary hippocampal neurons.Several individual members of the in total 22 Pcdhs-gamma were chosen to examine the expression of the three subfamilies, Pcdh-gamma A, -gamma B, and -gamma C. These Pcdh-gamma transcripts are expressed all over the brain, with minor regional and cell-type specific differences. Interestingly, a distinct, later onset of expression is observed for Pcdh-gamma C5, a gene located at the end of the Pedh-gamma cluster. Largely overlapping expression patterns of individual Pcdh-gamma proteins are detected with anti-peptide antibodies. Small differences are observed in the staining of dendritic processes and synapse-rich layers.Our results support the idea that Pcdhs-gamma participate in neuronal differentiation and may be implicated in the fine-tuning of neuronal morphology and synaptogenesis. Cell autonomous regulation of transcription might generate the widespread distribution of individual Pcdh-gamma in the brain, which is strikingly different from the restricted expression patterns observed for classical cadherins. Thus, a defined set of Pcdhs-gamma may engage in neuronal adhesion and signaling on the cellular level. (c) 2005 Elsevier Inc. All rights reserved.

The recently described protocadherin gene clusters encode cadherin-related proteins, which are highly expressed in the vertebrate nervous system. Here, we report biochemical studies addressing proteolytic processing of gamma-protocadherins. These type-I transmembrane proteins are cleaved by a metalloproteinase in vivo, generating a soluble extracellular fragment and a carboxyl-terminal fragment associated with the cellular membrane. In addition, we show that the carboxyl-terminal fragment is a substrate for further cleavage mediated by presenilin. Consequently, accumulation of the fragment is found when gamma-secretase is inactivated either by the specific presenilin-inhibitor L685,458 or in double mutant murine embryonic fibroblasts lacking both presenilin genes. The gamma-secretase-generated carboxyl-terminal fragment is largely unstable but accumulates when proteasomal degradation is inhibited. Interestingly, the proteolytic fragment generated by gamma-secretase can localize to the nucleus. This is the first report providing experimental evidence for a cell surface receptor signaling function of protocadherins regulated by proteolytic events.

The clustered protocadherins (Pcdhs) comprise >50 putative synaptic recognition molecules that are related to classical cadherins and highly expressed in the nervous system. Pcdhs are organized into three gene clusters (alpha, beta, and gamma). Within the alpha and gamma clusters, three exons encode the cytoplasmic domain for each Pcdh, making these domains identical within a cluster. Using an antibody to the Pcdh-gamma constant cytoplasmic domain, we find that all interneurons in cultured hippocampal neurons express high levels of Pcdh-gamma(s) in a nonsynaptic distribution. In contrast, only 48% of pyramidal-like cells expressed appreciable levels of these molecules. In these cells, Pcdh-gamma(s) were associated with a subset of excitatory synapses in which they may mediate presynaptic to postsynaptic recognition in concert with classical cadherins. Immunogold localization in hippocampal tissue showed Pcdh-gamma(s) at some synapses, in nonsynaptic plasma membranes, and in axonal and dendritic tubulovesicular structures, indicating that they may be exchanged among synapses and intracellular compartments. Our results show that although Pcdh-gamma(s) can be synaptic molecules, synapses form lacking Pcdh-gamma(s). Thus, Pcdh-gamma(s) and their relatives may be late additions to the classical cadherin-based synaptic adhesive scaffold; their presence in intracellular compartments suggests a role in modifying synaptic physiology or stability.

Protocadherins constitute the largest subgroup within the cadherin family of calcium-dependent cell-cell adhesion molecules. Recent progress in genome sequencing has enabled a refined phylogenetic analysis of protocadherins and led to the discovery of three large protocadherin clusters on human chromosome 5/mouse chromosome 18. Interestingly, many of the circa 70 protocadherins in mammals are highly expressed in the central nervous system. Roles in tissue morphogenesis and formation of neuronal circuits during early vertebrate development have been inferred. In the postnatal brain, protocadherins are possibly involved in the modulation of synaptic transmission and the generation of specific synaptic connections.

Myelin and lymphocyte protein (MAL) is a putative tetraspan proteolipid that is highly expressed by Schwann cells and oligodendrocytes as a component of compact myelin. Outside of the nervous system, MAL is found in apical membranes of epithelial cells, mainly in the kidney and stomach. Because MAL is associated with glycosphingolipids, it is thought to be involved in the organization, transport, and maintenance of glycosphingolipid-enriched membrane microdomains. In this report, we describe the generation and analysis of transgenic mice with increased MAL gene dosage. Immunohistochemical analysis revealed that the localization of MAL overexpression in the transgenic animals corresponded closely to the MAL expression pattern observed in wildtype animals, indicating correct spatial regulation of the transgene. Phenotypically, MAL overexpression led to progressive dissociation of unmyelinated axons from bundles in the PNS, a tendency to hypomyelination and aberrant myelin formation in the CNS, and the formation of large cysts in the tubular region of the kidney. Thus, increased expression of MAL appears to be deleterious to membranous structures in the affected tissues, indicating a requirement for tight control of endogenous MAL expression in Schwann cells, oligodendrocytes, and kidney epithelial cells.

Miniaturized interference filters were designed and fabricated using three different manufacturing technologies. Applying micro-milled ceramic masks during the coating processes different arrays of interference filters with 1 mm lateral feature size were arranged on a 3-inch Si-substrate. The spectral sensitivity of receiver cells has been modified by direct coating using ion assisted deposition (IAD) at low temperature. IAD-processes also allow the application of microlithographic masks, which are removable by organic solvents after the deposition process (lift-off). As an example three different miniaturized interference filters (RGB) were arranged side by side with lateral filter dimensions of a few tens of microns. A combination of coating processes, microlithographic masking procedures, and reactive ion etching (RIE) made it possible to arrange three different stripe filters with minimum filter features of about 5 mu m side by side.

High-speed sensors have been developed for industrial color and position analysis. These sensors consist of Si-PIN-diodes covered by miniaturized RGB-interference filters, a micro-lens array, an imaging micro-lens, and a glass cover plate. The basic color-receiver comprises three rhombic Si-PIN diodes arranged in a basic hexagonal pattern with a diameter of 700 mu m. To allow a simultaneous color and position control of still samples as well as for moving samples, e.g. samples lying on assembly lines or conveyor belts, the basic pattern has been arranged in two different receiver arrays. Both diode patterns each consisting of 64 diodes are arranged in user-defined arrays making efficient use of the available sensor area. The diodes are individually contacted to provide a rapid access to certain pixels or pixel clusters. Using interference filters the desired spectral band is transmitted to the detector almost without losses and the undesired band is blocked by reflection. The imaging microlens determines object distance, total visual field, and resolution. By using a coated glass cover plate the effective range is restricted to the desired spectral region. The object distance is adjustable between 10 mm and 50 mm and the diameter of the visual field is between 5 mm and 50 mm. The maximum variance in object height is 0.5 mm and the maximum lateral resolution is 0.5 mm x 0.5 mm. The operating frequency of the total sensor system depends on the data processing unit and is between 2 kHz and 10 kHz. These sensors make high-speed color analysis possible for a frequency range in which CCD-systems are too slow.

Two different types of color sensor have been developed, which are adaptable to a wide variety of industrial applications. One kind of sensor was developed by depositing dielectric interference filters directly on photoelectric cells, applying a low temperature coating procedure. This way it was possible to accommodate spectral characteristics to the specific spectral sensitivity of the photoelectric cells, minimizing losses by absorption and scattered light in the desired spectral region. The geometric shape of these coated cells is suitable especially for color measurements using fibers. The second kind of color sensors, consisting of miniaturized photoelectric cell arrays and three different micro-patterned interference filter arrays, arranged on a glass substrate, make it possible to distinguish and measure colors with a local resolution. The honey-comb-arrangement of filter- and receiver cells and a flexible setup also permits a fast adaptation to different applications. In this paper design and development of both miniaturized interference filers are described. The different manufacturing procedures are depicted, advantages and questions arising with the employment of interference filters are discussed and the entire assembly of both sensors is presented.

Diffraction efficiencies of binary dielectric multilayer gratings are calculated by combining thin film theory and scalar electromagnetic theory, and results are compared with efficiencies of conventional binary gratings, consisting of one single bulk material. By optimizing the number of layers and layer parameters such as material and layer thickness, multilayer grating designs are obtained showing diffraction efficiencies in the desired order close to the theoretical maximum and suppressing the zeroth diffraction order effectively. To meet technological requirements of etching processes such as a suitable etch-depth control, parameters of etch-stop layers are included into the calculation and optimization procedure. Manufacturing dielectric multilayer gratings including etch-stop layers will make it possible to obtain uniform etch profiles, a moderate surface roughness in the etched area, and high accuracies in etch depth without any online control devices. (C) 1998 Society of Photo-Optical Instrumentation Engineers. [S0091-3286(98)00106-8].

rMAL, the rat myelin and lymphocyte protein, is a small hydrophobic protein of 17 kDa with four putative transmembrane domains and is expressed in oligodendrocytes and Schwann cells, the myelinating cells of the nervous system. In addition, transcript expression has been found in kidney, spleen, and intestine. Confocal microscopy and immunoelectron microscopy with an affinity-purified antibody localized rMAL to compact myelin in a pattern similar to the structural myelin proteins: myelin basic protein and proteolipid protein. In kidney and stomach epithelia, rMAL is located almost exclusively on the apical (luminal) membranes of the cells lining distal tubuli in kidney and the glandular part of the stomach. Biochemical analysis of plasma membranes isolated from spinal cord and kidney demonstrated that rMAL is a proteolipid that is present in detergent insoluble complexes typical for proteins associated with glycosphingolipids. Lipid and protein analysis showed a co-enrichment of glycosphingolipids and rMAL protein within these complexes, indicating a close association of rMAL to glycosphingolipids in myelin and in kidney in vivo. We conclude that specific rMAL-glycosphingolipid interactions may lead to the formation and maintenance of stable protein-lipid microdomains in myelin and apical epithelial membranes. They may contribute to specific properties of these highly specialized plasma membranes.

Dielectric microfilters are designed and developed providing different filter functions on 3-in. substrates. Combining several coating procedures, microlithographic techniques, and dry-etching processes, spectral properties of basic filters are modified within patterns with feature sizes down to 5 mu m, resulting in large-area filter patterns (>5 cm(2)) with multiple spectral characteristics and vertical edge profiles. By applying these techniques within a three-step-iterative procedure red, green, and blue filters (RGB) are arranged edge to edge on one substrate to form a multispectral dielectric microfilter array for use as a key component in a local resolving color sensor. These microfilters are arranged in an array of 19 identical hexagons consisting of three rhombi each. The number of these arrays amounts to 81 on a single 3-in. substrate. A modular setup of a compact local resolving color sensor is presented, consisting of Si p-i-n photoelectric cells, dielectric microfilter arrays, an imaging lens, and a final glass cover plate with an IR-blocking filter. (C) 1998 Society of Photo-Optical instrumentation Engineers.

Micropatterned interference filters are developed by dry etching of dielectric multilayer stacks. The application of reactive-ion etching (RIE) techniques combined with the use of an etchstop layer are shown to be quite effective in producing two-stage micropatterns with defined spectral properties. By using a MgF2 underlayer the etching of TiO2 and SiO2 layers is stopped exactly, which provides the possibility to develop large area patterns (>1×1 cm2) with feature sizes as small as 10 μm. The distortion of edge features is shown to be better than 1 μm. A filter array was developed providing high-reflection and antireflection coatings within a period of 20 μm. © 1997 Society of Photo-Optical Instrumentation Engineers.

External modulators and switches for multimode fiber transmission systems are required for a plenty of applications in optical metrology and communication systems. We show that a confocal arrangement of microlens arrays with certain filter elements in the common focal plane of the arrays, which are moved with the help of piezoelectrical actuators, is a very simple and flexible concept to meet the needs of a number of very different applications. The focal length, lens pitch and width of the arrays have to be chosen properly. We discuss which optical elements (collimation optics, microlens arrays, focussing optics) are required for certain applications. We present experiments for an intensity modulator.

For specified colour measuring devices miniaturized dielectric filter arrays have been designed and developed. The manufacturing procedure is based on the combination of coating- and micropatterning processes including the employment of etchstop layers. The spectral properties of a basic filter, fabricated as an unpatterned filter blank, have been modified within small areas by Reactive ion Etching (RIE) a defined number of layers. This way dielectric interference filters with two different spectral characteristics have been arranged edge by edge within a period of 25 mu m.In this paper design and development of two-colour microfilter arrays are discribed and problems are depicted, arising with the production of multicolour dielectric microfilters. An alternative manufacturing procedure is dicussed, making it possible to arrange more than two different interference filters edge by edge.

Micropatterned interference filters have been developed by dry etching of dielectric multilayer stacks. Apart from the optical demands on the conventional filter design there are new requirements originating from the patterning process, which has to be stopped at the interface between two layers. In this paper the fundamental problems associated with the design and the development of micropatterned multilayer dielectric stacks are discussed. Details of the production of a two-stage filter array are described and experimental results are presented.