PURPOSE In experimental eye research, zebrafish has become a powerful model for human retina disorders. The purpose of the present study is the characterization of antibodies commonly employed in zebrafish models for rod photoreceptor degeneration. METHODS The 1D4 monoclonal antibody, developed against bovine rhodopsin, has been widely used in studies addressing structural and functional features of rhodopsin and was reported as an informative marker to stain rod outer segments in both mice and zebrafish. We have used transgenic reporter lines and histologic analysis to determine the photoreceptor types identified by 1D4 and other antibodies in zebrafish. RESULTS We demonstrate that 1D4, in contrast to what has been reported previously, does not recognize rod outer segments in zebrafish, but instead labels long double cone outer segments consistent with sequence conservation of the respective epitope. As an alternative marker for zebrafish rods, we characterized the monoclonal antibody zpr-3, which was found to stain outer segments of both rods, as well as double cones. CONCLUSIONS Our findings highlight the importance to confirm specificity of antibodies in cross-species experiments for correct interpretation of experimental data. Our findings clarify conflicting published information arising from studies using 1D4 and zpr-3 antibodies in zebrafish.

Background Retinitis pigmentosa (RP) is an inherited eye disease characterized by the progressive degeneration of rod photoreceptor cells. Mutations in pre-mRNA splicing factors including PRPF31 have been identified as cause for RP, raising the question how mutations in general factors lead to tissue specific defects. Results We have recently shown that the zebrafish serves as an excellent model allowing the recapitulation of key events of RP. Here we use this model to investigate two pathogenic mutations in PRPF31 , SP117 and AD5, causing the autosomal dominant form of RP. We show that SP117 leads to an unstable protein that is mislocalized to the rod cytoplasm. Importantly, its overexpression does not result in photoreceptor degeneration suggesting haploinsufficiency as the underlying cause in human RP patients carrying SP117. In contrast, overexpression of AD5 results in embryonic lethality, which can be rescued by wild-type Prpf31. Transgenic retina-specific expression of AD5 reveals that stable AD5 protein is initially localized in the nucleus but later found in the cytoplasm concurrent with progressing rod outer segment degeneration and apoptosis. Importantly, we show for the first time in vivo that retinal transcripts are wrongly spliced in adult transgenic retinas expressing AD5 and exhibiting increased apoptosis in rod photoreceptors. Conclusion Our data suggest that distinct mutations in Prpf31 can lead to photoreceptor degeneration through different mechanisms, by haploinsufficiency or dominant-negative effects. Analyzing the AD5 effects in our animal model in vivo , our data imply that aberrant splicing of distinct retinal transcripts contributes to the observed retina defects.

Background High mobility group A (HMGA) proteins regulate gene transcription through architectural modulation of chromatin and the formation of multi-protein complexes on promoter/enhancer regions. Differential expression of HMGA variants has been found to be important for distinct differentiation processes and deregulated expression was linked to several disorders. Here we used mouse C2C12 myoblasts and C2C12 cells stably over-expressing HMGA1a-eGFP to study the impact of deregulated HMGA1 expression levels on cellular differentiation. Results We found that induction of the myogenic or osteogenic program of C2C12 cells caused an immediate down-regulation of HMGA1. In contrast to wild type C2C12 cells, an engineered cell line with stable over-expression of HMGA1a-eGFP failed to differentiate into myotubes. Immunolocalization studies demonstrated that sustained HMGA1a-eGFP expression prevented myotube formation and chromatin reorganization that normally accompanies differentiation. Western Blot analyses showed that elevated HMGA1a-eGFP levels affected chromatin composition through either down-regulation of histone H1 or premature expression of MeCP2. RT-PCR analyses further revealed that sustained HMGA1a expression also affected myogenic gene expression and caused either down-regulation of genes such as MyoD, myogenin, Igf1 , Igf2 , Igfbp1-3 or up-regulation of the transcriptional repressor Msx1 . Interestingly, siRNA experiments demonstrated that knock-down of HMGA1a was required and sufficient to reactivate the myogenic program in induced HMGA1a over-expressing cells. Conclusions Our data demonstrate that HMGA1 down-regulation after induction is required to initiate the myogenic program in C2C12 cells. Sustained HMGA1a expression after induction prevents expression of key myogenic factors. This may be due to specific gene regulation and/or global effects on chromatin. Our data further corroborate that altered HMGA1 levels influence the expression of other chromatin proteins. Thus, HMGA1 is able to establish a specific chromatin composition. This work contributes to the understanding of how differential HMGA1 expression is involved in chromatin organization during cellular differentiation processes and it may help to comprehend effects of HMGA1 over-expression occurring in malign or benign tumours.

Retinitis pigmentosa (RP) is a common hereditary eye disease that causes blindness due to a progressive loss of photoreceptors in the retina. RP can be elicited by mutations that affect the tri-snRNP subunit of the pre-mRNA splicing machinery, but how defects in this essential macromolecular complex transform into a photoreceptor-specific phenotype is unknown. We have modeled the disease in zebrafish by silencing the RP-associated splicing factor Prpf31 and observed detrimental effects on visual function and photoreceptor morphology. Despite reducing the level of a constitutive splicing factor, no general defects in gene expression were found. Instead, retinal genes were selectively affected, providing the first in vivo link between mutations in splicing factors and the RP phenotype. Silencing of Prpf4, a splicing factor hitherto unrelated to RP, evoked the same defects in vision, photoreceptor morphology and retinal gene expression. Hence, various routes affecting the tri-snRNP can elicit tissue-specific gene expression defects and lead to the RP phenotype.

Eukaryotic cells devoid of mitochondrial DNA (rho0 cells) were originally generated under artificial growth conditions utilizing ethidium bromide. The chemical is known to intercalate preferentially with the mitochondrial double-stranded DNA thereby interfering with enzymes of the replication machinery. Rho0 cell lines are highly valuable tools to study human mitochondrial disorders because they can be utilized in cytoplasmic transfer experiments. However, mutagenic effects of ethidium bromide onto the nuclear DNA cannot be excluded. To foreclose this mutagenic character during the development of rho0 cell lines, we developed an extremely mild, reliable and timesaving method to generate rho0 cell lines within 3-5 days based on an enzymatic approach. Utilizing the genes for the restriction endonuclease EcoRI and the fluorescent protein EGFP that were fused to a mitochondrial targeting sequence, we developed a CMV-driven expression vector that allowed the temporal expression of the resulting fusion enzyme in eukaryotic cells. Applied on the human cell line 143B.TK- the active protein localized to mitochondria and induced the complete destruction of endogenous mtDNA. Mouse and rat rho0 cell lines were also successfully created with this approach. Furthermore, the newly established 143B.TK- rho0 cell line was characterized in great detail thereby releasing interesting insights into the morphology and ultra structure of human rho0 mitochondria.

In all eukaryotic cells, origins of DNA replication are characterized by the binding of the origin recognition complex (ORC). How ORC is positioned to sites where replication initiates is unknown, because metazoan ORC binds DNA without apparent sequence specificity. Thus, additional factors might be involved in ORC positioning. Our experiments indicate that a family member of the high-mobility group proteins, HMGA1a, can specifically target ORC to DNA. Coimmunoprecipitations and imaging studies demonstrate that HMGA1a interacts with different ORC subunits in vitro and in vivo . This interaction occurs mainly in AT-rich heterochromatic regions to which HMGA1a localizes. Fusion proteins of HMGA1a and the DNA-binding domain of the viral factor EBNA1 or the prokaryotic tetracycline repressor, TetR, can recruit ORC to cognate operator sites forming functional origins of DNA replication. When HMGA1a is targeted to plasmid DNA, the prereplicative complex is assembled during G 1 and the amount of ORC correlates with the local concentration of HMGA1a. Nascent-strand abundance assays demonstrate that DNA replication initiates at or near HMGA1a-rich sites. Our experiments indicate that chromatin proteins can target ORC to DNA, suggesting they might specify origins of DNA replication in metazoan cells.

Extensive epidemiological data indicate that inorganic arsenic is associated with several types of human cancer. Nevertheless, the underlying mechanisms are poorly understood. Among its mode of action are the alterations on DNA methylation, which provoke aberrant gene expression. However, beyond DNA methylation, little is known about arsenic’s effects on chromatin. In this study, we investigated the effects of sodium arsenite (NaAsO 2 ) on global histone modifications and nucleosome-associated proteins. Our findings revealed that NaAsO 2 exposure significantly increases global histone acetylation. This effect was related to the inhibition of histone deacetylase (HDAC) activity because NaAsO 2 was able to inhibit HDACs comparable to the well-known HDAC inhibitor trichostatin A (TSA). Furthermore, analyses of the dynamic properties of the nucleosome-associated high mobility group N proteins demonstrate that NaAsO 2 elevates their mobility. Thus, our data suggest that NaAsO 2 induces chromatin opening by histone hyperacetylation due to HDAC inhibition and increase of the mobility of nucleosome-associated proteins. As the chromatin compaction is crucial for the regulation of gene expression as well as for genome stability, we propose that chromatin opening by NaAsO 2 may play a significant role to impart its genotoxic effects.

The high mobility group proteins A2 (HMGA2) have been implicated in the control of cell proliferation and differentiation, in particular during embryogenesis. Here, we used Xenopus laevis to analyze HMGA2 gene expression patterns during oogenesis and early embryogenesis. We found two functional XlHMGA2 isoforms, which we named XlHMGA2α and XlHMGA2β. As revealed by RT-PCR, real-time PCR and whole-mount in situ hybridization both mRNAs are maternally produced and stored in eggs. Whole-mount in situ hybridizations revealed a conspicuous redistribution of the XlHMGA2 transcripts during early embryogenesis. Initially, during oogenesis and in eggs, the transcripts are uniformly distributed in the cytoplasm. With activation of the eggs the transcripts accumulate near the animal pole and remain in the juxtanuclear regions of animal pole blastomeres until midblastula transition. According to real-time PCR data, XlHMGA2α appears to be preferentially expressed during oogenesis and after midblastula transition, whereas XlHMGA2β expression predominates after neurulation, suggesting an individual transcriptional regulation.

Durch die Kombination dieser drei Untersuchungsgruppen ließen sich zweifelsohne wichtige Einblicke gewinnen. Vor allem gestattete sie es aber, das bunte Spektrum der Dysostosen besser zu würdigen. Glaubte man nämlich noch vor 20 Jahren, nur je einen gut umschriebenen Typus, die Hunter-P faundler-Hurler sowie dié Morquio-Brailsfordsche Dysostose, unterscheiden zu müssen, so ließen sich doch in der ersten Gruppe middestens sechs Formen, in der zweiten mindestens vier unterschiedliche Krankheitsbilder individualisieren. Mit großer Wahrscheinlichkeit werden in den kommenden Jahren noch weitere Typen hinzukommen (als ein Beispiel einer Sonderform sei der Tarzan-Typus Giedion angeführt). Nur eine gezielte, individualisierende Analyse kann den Einblick in den bisher noch verwirrenden Formenkreis der Dysostosen fördern. Sie Ist jedenfalls vielversprechender als die übliche Einfügung in die enchoncirale Dysostose", diesen nichtssagenden Namen, wie Lenz sich ausdrückt. Im Sinne einer analytischen Betrachtung sei die nachfolgende Beobachtung mitgeteilt, deren klinisches Bild zunächst an das Vorliegen einer Morquio-Brails/ordschen Dysostose denken ließ.

Beschreibung der im Röntgenbild nachweisbaren, zum Teil sehr auffallenden Knochenveränderungen in einer Familie, in welcher fünf Befallene in zwei Generationen an einem Geroderma osteodysplastica hereditaria leiden. Der Erbgang dieser nosologisch autonomen, von der Progerie unabhängigen Affektion ist geschlechtsgebunden mit inkompletter Dominanz. Auf die Bedeutung der Röntgenuntersuchung der Wirbelsäule, welche sehr charakteristische Befunde zutage fördert, wird besonders verwiesen.

Beschreibung eines Berstungsbruches des Atlas nach Jefferson, kombiniert mit einer atlanto-okzipitalen Luxation nach vorn. Der Nachweis dieser Verletzungen erfolgt vorwiegend im Schichtbild.

Besprechung der Wirbelbogenveränderungen: a) in der Bogenwurzel (Verkürzung, Verdickung, retrosomatische Spalte); b) im Zwischengelenksstück (Verlängerung, Knickung, Verschmälerung, abnorme Struktur, Unterbrechungslinie), c) in der Lamina (mediane und retroisthmische Spalte, Hypoplasie, Aplasie, Verschmelzungen).

Dysplasie des Basioccipitale und beider Exoccipitalia. Schweres ponto-zerebellares Krankheitsbild. Möglichkeit der Diagnose am Lebenden mittels der Schichtuntersuchung im frontalen und sagittalen Strahlengang.