Please be advised that this information was generated on 2017-03-31 and may be subject to change. In order to elucidate the cellular and molecular processes which are involved in Norrie disease (NO), we have used gene targeting technology to generate ND mutant mice. The murine homologue of the ND gene was cloned and shown to encode a polypeptide that shares 94% of the amino acid sequence with its human counterpart. RNA in situ hybridization revealed expression in retina, brain and the olfactory bulb and epithelium of 2 week old mice. Hemizygous mice carrying a replacement mutation in exon 2 of the ND gene developed retrolental structures in the vitreous body and showed an overall disorganization of the retinal ganglion cell layer. The outer plexiform layer disappears occasionally, resulting in a juxtaposed inner and outer nuclear layer. At the same regions, the outer segments of the photoreceptor cell layer are no longer present. These ocular findings are consistent with observations in ND patients and the generated mouse line provides a faithful model for study of early pathogenic events in this severe X-linked recessive neurological disorder.

Screening of a human brain cDNA library using the C-terminal tail of the melanin-concentrating hormone receptor 1 (MCHR1) as bait in a yeast two-hybrid assay resulted in the identification of the neurite-outgrowth related factor, neurochondrin. This interaction was verified in overlay, pulldown, and co-immunoprecipitation assays. Deletion mapping confined the binding to the C terminus of neurochondrin and to the proximal C terminus of MCHR1, a region known to be involved in G protein binding and signal transduction. This region of the MCHR1 is also able to interact with the actin- and intermediate filament-binding protein, periplakin. Interactions of MCHR1 with neurochondrin and periplakin were competitive, indicating that these two proteins bind to overlapping regions of MCHR1. Although neurochondrin did not interfere with melanin-concentrating hormone-mediated internalization of the receptor, it did inhibit G protein-coupled signal transduction via both Gαi/o and Gαq/11 family G proteins as measured by each of melanin-concentrating hormone-induced G protein-activated inwardly rectifying K+ channel activity of voltage-clamped amphibian oocytes, by calcium mobilization in transfected mammalian cells, and by reduction in the capacity of melanin-concentrating hormone to promote binding of [35S]guanosine 5′-3-O-(thio)triphosphate to both Gαo1 and Gα11. Immunohistochemistry revealed co-expression of neurochondrin and MCHR1 within the rodent brain, suggesting that neurochondrin may be involved in the regulation of MCHR1 signaling and play a role in modulating melanin-concentrating hormone-mediated functions in vivo.

We have recently identified a Melanin-concentrating hormone receptor 1 interacting zinc-finger protein (MIZIP) from a human brain cDNA library. Here, we report the generation of a specific antibody against MIZIP and its distribution in rodent tissues using immunoblotting and immunohistochemical techniques. MIZIP was detected as a 27 kDa protein in brain, liver, and skeletal muscle, and to a lower extend, in lung, testis, and heart. Subcellular fractionation of adult mouse brain revealed the presence of MIZIP and MCHR1 in the cytoplasmic, membrane, and synaptosomal fraction, but not in a postsynaptic density preparation. In cultured rat, embryonic hippocampal neurons MIZIP is somatodendritically localized. In the adult rodent brain, MIZIP is widely distributed. High levels of expression were detected in brain regions involved in olfaction, feeding behavior, sensorimotor integration, and learning and memory, for example, the olfactory bulb, the olfactory tubercle, the caudate putamen, the thalamus and hypothalamus, the nucleus accumbens, the cerebral cortex, the hippocampus formation, and the cerebellum. Co-expression of MIZIP and MCHR1 was observed, for example, in pyramidal neurons of the cerebral cortex and hippocampus, in neurons of the olivary nucleus, lateral hypothalamus, nucleus accumbens, caudate putamen, pontine, and mesencephalic trigeminal nucleus. However, there are also differences in the expression patterns, for example, high expression of MCHR1 was detected in the lateral habenula, but no expression of MIZIP. These data support the notion that MIZIP might interact with MCHR1 in a cell type specific manner in vivo, suggesting a role in the regulation of MCH signalling in distinct regions of the mammalian brain.

MIZIP was originally identified as a highly conserved zinc-finger protein from human brain interacting with the C-terminus of the melanin-concentrating hormone receptor 1. However, the cellular functions of MIZIP are still not known. Here, we focussed on the identification of associated proteins using affinity purification from human cells. This resulted in the identification of alpha- and beta-tubulin. The interaction was confirmed in vitro and in vivo using GST pull-down and immunoprecipitation assays, and was mapped to the MYND zinc-finger of MIZIP and to the N-terminus of tubulin. Immunoprecipitation and immunocytochemistry analyses demonstrate that MIZIP binds to tubulin but not to cellular microtubules in vivo and that ectopic expression of MIZIP does not interfere with the overall structure of the microtubular cytoskeleton. Our results suggest that MIZIP might play an important role in mammalian cells by associating with tubulin and thus might provide a link between MCHR1 and tubulin functions.

Using the yeast‐two‐hybrid system a novel protein was identified from human brain that interacts with the C‐terminus of melanin‐concentrating hormone receptor 1 (MCH‐R1). This protein, characterized by a Myeloid translocation protein 8, Nervy, DEAF1 proteins (MYND) zinc‐finger domain, is termed MCH‐R1‐interacting zinc‐finger protein, MIZIP. It is fully conserved in man, rat, mouse and highly conserved in Xenopus and zebrafish, but not detectable in invertebrates. MIZIP gene organization in human (six exons on chromosome 9q34.3) and mouse is highly conserved, yet in rodents an additional exon is generated giving rise to alternatively spliced mRNAs. MIZIP is expressed in brain, testis and stomach, where expression of MCH and MCH‐R1 was previously reported. MIZIP interaction with MCH‐R1 was verified by overlay and pull‐down assays as well as by co‐transfection experiments in human embryonic kidney‐293 cells. MIZIP is cytoplasmically localized but gets recruted to the plasma membrane when cells are co‐transfected with MCH‐R1 supporting the notion that MIZIP is involved in the function of MCH‐R1.

We have isolated and characterized a novel differentially spliced gene predominantly expressed in the nervous system, which encodes protein isoforms with significant homology to the alpha-actinin protein superfamily, the Caenorhabditis elegans UNC-53 protein and weak homology to the nuclear membrane protein POM121. Similar to POM121 the primary structures show a hydrophobic region that is likely to form one or more adjacent transmembrane segment(s). Indirect immunofluorescence with antibodies against a synthetic peptide gave staining of the nucleus. Target experiments with EGFP (enhanced green fluorescent protein)-fusion proteins confirmed the nuclear localization. Two further members of this gene family could be isolated. All three pore membrane and/or filament interacting like (POMFIL) genes are differentially expressed in neuronal tumor cell lines. In 40% of tested primary neuroblastomas expression of POMFIL1 is strongly reduced and after brain injury POMFIL1 protein expression is upregulated, indicating that POMFIL1 is involved in the process of neuron growth and regeneration, as well as in neural tumorigenesis.

The BMP2-dependent onset of osteo/chondrogenic differentiation in the acknowledged pluripotent murine mesenchymal stem cell line (C3H10T1/2) is accompanied by the immediate upregulation of Fibroblast Growth Factor Receptor 3 (FGFR3) and a delayed response by FGFR2. Forced expression of FGFR3 in C3H10T1/2 is sufficient for chondrogenic differentiation, indicating an important role for FGF-signaling during the manifestation of the chondrogenic lineage in this cell line. Screening for transcription factors exhibiting a chondrogenic capacity in C3H10T1/2 identified that the T-box containing transcription factor Brachyury is upregulated by FGFR3-mediated signaling. Forced expression of Brachyury in C3H10T1/2 was sufficient for differentiation into the chondrogenic lineage in vitro and in vivo after transplantation into muscle. A dominant-negative variant of Brachyury, consisting of its DNA-binding domain (T-box), interferes with BMP2-mediated cartilage formation. These studies indicate that BMP-initiated FGF-signaling induces a novel type of transcription factor for the onset of chondrogenesis in a mesenchymal stem cell line. A potential role for this T-box factor in skeletogenesis is further delineated from its expression profile in various skeletal elements such as intervertebral disks and the limb bud at late stages (18.5 d.p.c.) of murine embryonic development.

The murine homologue of the human glycoprotein (transmembrane) NMB (GPNMB) gene was identified by subtractive cloning from in vitro cultured murine primary osteoblast cells and subsequent RACE-PCR. GPNMB is a highly glycosylated type I transmembrane protein that shares significant sequence homology to several melanosomal proteins. Increasing expression of Gpnmb mRNA was observed during differentiation of murine primary osteoblast cell cultures. To address the potential functions of GPNMB we analysed its mRNA-expression during murine embryonic development. In early development Gpnmb mRNA is detected at high levels in the outer layer of the retina. Later in development expression gets restricted to the retinal pigment epithelium and iris. At the cytoplasmic domain of GPNMB, a conserved di-leucin-based endosomal/melanosomal-sorting signal (ExxPLL) was located, present as well in several known melanosomal proteins. To analyse the subcellular localization we used EGFP-tagged GPNMB transfected in COS7 and HEK293 cells. In both non-pigmented cell lines, the EGFP–GPNMB fusion protein was localized to vesicular, endosomal like structures. Sequence homology to known melanosomal proteins, mRNA expression and subcellular localization are suggestive for GPNMB as an intracellular, endosomal/melanosomal compartment specific protein important for melanin biosynthesis and the development of the retinal pigment epithelium and iris. As the gene coding for human GPNMB was localized to chromosome 7p15, a locus involved in the human inherited disease cystoid macular edema, also known as dominant cystoid macular dystrophy (OMIM 153880) we highly suggest that GPNMB is a candidate gene for this human inherited disease.

62 The species and chromosomal distribution of the centromeric -satellite I sequence from sheep in the tribe Caprini and other Bovidae Chaves R, Guedes-Pinto H, Heslop-Harrison JS, Schwarzacher T 67 TSPY variants in six loci on the human Y chromosome Dechend F, Williams G, Skawran B, Schubert S, Krawczak M, Tyler-Smith C, Schmidtke J 72 Local mechanisms in sex specific morphogenesis Drews U 81 Microdeletion 4p16.3 in three unrelated patients with Wolf-Hirschhorn syndrome Dufke A, Seidel J, Schöning M, Döbler-Neumann M, Kelbova C, Liehr T, Beensen V, Backsch C, Klein-Vogler U, Enders H 85 A second case of inv(4)pat with both recombinants in the offspring: rec dup(4q) in a girl with Wolf-Hirschhorn syndrome and rec dup(4p) Dufke A, Eggermann K, Balg S, Stengel-Rutkowski S, Enders H, Kaiser P

For five members of the family of the small leucine‐rich proteoglycans (SLRPs), the expression pattern during fetal development was analyzed. RNA in situ hybridization on whole body sections of mouse embryos was performed for biglycan (Bgn), decorin (Dcn), fibromodulin (Fmod), chondroadherin (Chad), and lumican (Lum). Special attention was given to the question of whether these patterns coincide only with sites of collagen secretion in connective tissue during tissue modeling or if expression can be observed at specific sites of organ differentiation also. In general, Fmod, Lum, and Bgn are expressed at sites of cartilage and bone formation and interstitial tissue deposition; Chad is expressed only at sites of cartilage; and Dcn is expressed only at sites of interstitial tissue deposition. However, there are some distinct developmental stages where no collagen secretion is known to occur. For example, this applies for the expression of Fmod in the forming somites of stage 9.5 postconception (p.c.), for Dcn and Lum in later stage embryos in the pituitary gland and dorsal root ganglia, and for Bgn and Dcn during differentiation in the kidney. These studies provide further evidence for a role of these molecules during connective tissue organization but also for an involvement at specific sites of organ differentiation.

Mutations in the DKC1 gene are responsible for causing X-linked recessive dyskeratosis congenita (DKC) and a more severe allelic variant of the disease, Hoyeraal-Hreidarsson syndrome. Both diseases are characterized by progressive and fatal bone marrow failure. The nucleolar protein dyskerin is the pseudouridine synthase component of the box H+ACA snoRNAs and also interacts with the RNA component (human telomerase, hTR) of the telomerase complex. Dyskerin is therefore thought to function in the processing of pre-rRNA and of the hTR, strengthening the notion that the underlying mechanism of DKC is a premature senescence of cells, especially of the rapidly dividing epithelial and hemopoietic cells. To examine the functions of dyskerin in vivo, it will be necessary to generate mouse models. As a first step, we here provide the genomic structure of the mouse Dkc1 gene and expression analysis of the transcript. Northern hybridizations revealed the tissue-specific expression of an alternative 4.5-kb transcript, in addition to the ubiquitous 2.6-kb transcript. RNA in situ hybridizations on day 10.5-18.5 postconception embryos showed a ubiquitous expression of Dkc1 with a notably higher level of expression confined to the epithelial tissues. In addition, higher level Dkc1 expression was confined to embryonic neural tissues as well as to specific neurons in the cerebellum (Purkinje cells) and the olfactory bulb (mitral cells) of the adult brain. In adult testis, elevated expression was limited to the Leydig cells. The results indicate that some of the pertinent functions of dyskerin may be more tissue-specific than previously thought and are not limited to rapidly dividing cells.