Antisense targeting of oxytocinergic hypothalamus neurons induces cytoplasmic triple helix-like immunoreactivity.

Intracerebroventricular injections of oligonucleotide probes complementary to oxytocin mRNA are known to decrease systemic oxytocin levels. In this study we show that immunoreactive oxytocin in the magnocellular hypothalamic perikarya and in their neurohypophysial projections remains unaffected by intracerebroventricular injections with an oxytocin antisense probe in rats. Hybridization signal for oxytocin mRNA was increased in the supraoptic and paraventricular nuclei in these animals. Immunocytochemistry with a monoclonal antibody, raised against triple helical DNA resulted in an accumulation of cytoplasmic reaction product in many of the magnocellular oxytocin immunoreactive neurons and in a fraction of the Herring bodies inthe posterior pituitary lobe in the antisense treated rats. Such immunostaining could be abolished by pretreating sections with RNase H. Animals injected with a mismatch probe instead of the antisense probe were devoid of cytoplasmic or axonal triple helix immunostaining. Our findings indicate that oxytocinergic transcripts in magnocellular hypothalamic neurons form triple helix-like aggregates upon specific antisense targeting rather than being degraded by endogenous RNases. While de novo transcription of oxytocin is probably stimulated, systemic release of the nonapeptide may be impaired.

Reduced formamide content and hybridization temperature results in increased non-radioactive mRNA in situ hybridization signals.

To define conditions for highly sensitive non-radioactive mRNA in situ hybridization on cryostat sections the influence of decreased formamide content and hybridization temperature was studied. The examination was performed on fibromatosis nodules of palmar fibromatosis visualizing the beta actin mRNA of myofibroblasts. The results show that a decrease in formamide content and hybridization temperature is able to enhance the sensitivity of mRNA detection applicating digoxigenin labelled DNA oligodeoxynucleotide. The best hybridization signal could be obtained under formamide-free conditions. In conclusion, a simplified sensitive formamide-free mRNA in situ hybridization protocol using oligonucleotide probes on human tissue cryostat sections is presented. The negative formamide effect is seen as a result of the chemical interaction of formamide with nucleic acid strands. An omission of formamide is suggested if the target as well as the probe are single stranded.

Transcription specific differences visualized by fluorescence in situ hybridization pattern on interphase nuclei of different cell types.

Application of a "formamide free" and thus "material preserving" in situ hybridization technique using the cDNA of the myf3 gene revealed the following results: Human rhabdomyosarcoma cells, characterized by a high expression of myf3 show intensive hybridization signals in their interphase. RNase treatment prior to hybridization considerably reduces the size of this signals. In comparison, isolated nuclei of human lymphocytes in which no need for the expression of this gene exists, show barely hybridization signals. Correspondingly, RNase treatment had no effect on hybridization pattern at all. In conclusion an increased transcription efficiency of a cell type specific gene is accompanied by a higher hybridization accessibility in the corresponding cell nuclei.

TGF beta and bFGF synthesis and localization in Dupuytren's disease (nodular palmar fibromatosis) relative to cellular activity, myofibroblast phenotype and oncofetal variants of fibronectin.

Nodular palmar fibromatosis is a self-limited proliferation of fibro-/myofibroblasts associated with growth factor synthesis and abundant fibronectin extracellular matrix deposition. bFGF and TGF beta are potent modulators of fibro-/myofibroblast proliferation and differentiation. Moreover, in vitro investigations evidenced a TGF beta 1-dependent regulation of alternative splicing of fibronectin mRNA. To investigate a possible implication of these growth factors in the tissue formation process of palmar fibromatosis, TGF beta 1/2 and bFGF synthesis, as well as TGF beta 1/3 and bFGF tissue distribution, is demonstrated by RNA in situ hybridization and/or immunohistochemistry in relation to myofibroblast phenotype development (alpha-smooth muscle actin, desmin immunohistochemistry), expression of different fibronectin isoforms (ED-A+, ED-B+ and oncofetal glycosylated fibronectin immunohistochemistry, fibronectin RNA in situ hybridization) and cellular activity (cyclin RNA in situ hybridization, Ki-67 immunolabelling). The myofibroblast phenotype (alpha-smooth muscle actin, desmin), the growth factor synthesis (TGF beta 1 and 2, bFGF), fibronectin matrix synthesis (RNA in situ hybridization with cDNA) and ED-A+, ED-B+ and oncofetal glycosylated fibronectin immunostaining are exclusively localized in the active proliferative nodules (Ki-67 immunolabelling and cyclin mRNA demonstration). Whereas the growth factor synthesis is restricted to the proliferative areas of the fibromatosis only, TGF beta 1, TGF beta 3 and bFGF proteins can also be detected immunohistochemically with a lower intensity in the surrounding aponeurotic tissue. The spatial correlation of myofibroblast phenotype, TGF beta and bFGF synthesis and the occurrence of the oncofetal molecular fibronectin variants (ED-B+ and oncofetal glycosylated fibronectin) in the active proliferative fibromatosis nodules suggests a pathogentic role of these growth factors and matrix components in the tumorous tissue formation process. The presence of the bFGF and TGF beta 1/3 proteins in fibroblasts neighbouring the proliferative nodules may point to a recruitment of quiescent aponeurotic fibroblasts in the fibromatous tissue formation process.

Differential expression of fibronectin splice variants, oncofetal glycosylated fibronectin and laminin isoforms in nodular palmar fibromatosis.

The tissue formation process in nodular palmar fibromatosis (Morbus Dupuytren) was investigated by the demonstration of fibronectin splice variants (ED-A and ED-B fibronectin), de novo glycosylated fibronectin and laminin isoforms (A, M, B1, B2, s chains) in association to the proliferative activity (Ki-67 antigen) and the occurrence of myofibroblast phenotype (alpha-smooth muscle actin, desmin). The proliferative noduli of the fibromatosis were characterized by a diffuse immunostaining for alpha-smooth muscle actin, and single cells positive for desmin and the Ki-67 antigen. In contrast to the surrounding aponeurosis as extracellular matrix, components of the whole proliferative noduli were defined: ED-A, ED-B and de novo glycosylated fibronectin, B1 and B2 laminin chain, tenascin and collagen type IV. The demonstration of the A and M laminin chain was restricted to a few cells of the proliferative noduli. S laminin could be visualized in the majority of palmar aponeurotic fibroblasts. As revealed by mRNA, in situ hybridization a de novo synthesis of fibronectin could only be detected within proliferative noduli. There is a positive correlation between the myofibroblast phenotype formation, cellular proliferation and the occurrence of ED-A and ED-B containing fibronectin, as well as de novo glycosylated fibronectin in Dupuytren's disease. The ultrastructural irregularities of myofibroblastic basal lamina and the heterogeneity of the myofibroblast phenotype are equivalent to the variability of laminin isoform immunostaining.

Sense- and antisense-targeting of oxytocinergic systems in the rat hypothalamus.

3' phosphorothioate modified sense or antisense oligonucleotides to oxytocin transcripts were used for in vivo targeting of oxytocin (OT) neurons in the rat hypothalamus. Intracerebroventricular injections of antisense probe resulted in a loss of systemic OT. However, abundant immunoreactive OT as well as oxytocin mRNA hybridization signal was visualized in the hypothalamo neurohypophysial system (HNS) of these animals. RT-PCR of hypothalamic homogenates revealed clearly detectable amounts of cytoplasmic OT mRNA in spite of sense or antisense treatment. Immunostaining with an antibody to DNA-RNA triple helix resulted in cytoplasmic reaction product in the HNS in the antisense group, which was not found when tissue sections had been pretreated with RNase. Animals injected with the sense probe showed a less pronounced but significant loss of systemic OT while immunoreactivity for this peptide in the posterior lobe seemed to be unaffected. RT-PCR of OT encoding mRNA extracted from sense injected rats indicated that these transcripts were of smaller size than samples from antisense treated animals or controls. Immunostaining with the triple helix antibody revealed distinct immunoreactive dots in cellular nuclei throughout the brain in the sense group. Our findings suggest that sense and antisense probes may not readily be employed as "functional antagonists" since peptidergic neurons are probably capable of responding in various ways to the treatment. RNase H may be less important in hypothalamic neurons as commonly suggested. Targeted transcripts are likely to form complexes which may somehow interact with secretion. Triple helix formation in the nucleus may not be able to induce an efficient transcriptional arrest. Although endocrine and behavioral changes observed in antisense treated animals seem to confirm the hypothesis that a selective translational "knock out" can be achieved with in vivo hybridization strategies, the actual underlying molecular events are far from being understood. On the other hand, sense or antisense strategies may provide valuable insights into molecular and cellular events associated with neurosecretion.

Rapid fluorescence in situ hybridization with repetitive DNA probes: quantification by digital image analysis.

Fluorescence in situ hybridization (FISH) has become an important tool not only in cytogenetic research but also in routine clinical chromosome diagnostics. Here, results of a quantification of fluorescence signals after in situ hybridization with repetitive DNA probes are reported using a non-enzymatic hybridization technique working with a buffer system not containing any formamide or equivalent chemical denaturing agents. Following simultaneous denaturation of both cells and DNA probes, the renaturation time was reduced to less than 30 min. For one of the DNA probes reasonable FISH-signals were even achieved after about 30 s renaturation time. In addition, the number of washing steps was reduced drastically. As a model system, two repetitive DNA probes (pUC 1.77, D15Z1) were hybridized to human metaphase spreads and interphase nuclei obtained from peripheral blood lymphocytes. The probes were labelled with digoxigenin and detected by FITC-anti-digoxigenin. The hybridization time was reduced step by step and the resulting fluorescence signals were examined systematically. For comparison the pUC 1.77 probe was also hybridized according to a FISH protocol containing 50% formamide. By renaturation for 2 h and overnight two FISH signals per nucleus were obtained. Using shorter renaturation times, no detectable FISH signals were observed. Quantification of the FISH signals was performed using a fluorescence microscope equipped with a cooled colour charge coupled device (CCD) camera. Image analysis was made interactively using a commercially available software package running on a PC (80486). For the pUC 1.77 probe the major binding sites (presumptive chromosomes 1) were clearly distinguished from the minor binding sites by means of the integrated fluorescence intensity. For the two (pUC 1.77) or four (D15Z1) brightest spots on the metaphase spreads and in the interphase nuclei hybridized without formamide, integrated fluorescence intensity distributions were measured for different renaturation times (0.5, 15, 30 min). The intra-nuclear variation in the intensity of the two brightest in situ hybridization spots appeared to be slightly higher (CV between 16 and 32%) than the corresponding variation in the metaphase spreads (CV between 10 and 19%). For the D15Z1 probe FISH signals were detected after hybridization without formamide and 15 min and 30 min renaturation. Always four bright spots were visible and tentatively assigned on the metaphase spreads (presumptive chromosome 15 and 9). The intensity variation of each pair of homologues in a metaphase spread showed a CV of 14 or 15%, respectively, for the presumptive chromosome 15, and 8 or 9%, respectively, for the presumptive chromosome 9.

A rapid FISH technique for quantitative microscopy.

Results of quantitative microscopy for fluorescence in situ hybridization (FISH) signals with repetitive DNA probes (pUC 1.77 and D15Z1) are reported. A nonenzymatic hybridization technique was applied using fluorescein-12-dUTP labeled DNA probes and a buffer system not containing any formamide or equivalent chemical denaturing agents. Following thermal denaturation, the renaturation time was reduced to less than 30 min. The number of wash steps was reduced to one. For the pUC 1.77 probe, the major binding sites (chromosome 1) were distinguished from the minor binding sites by means of fluorescence intensity and spot size. The intensity variation of the two brightest FISH spots (major binding sites) in the same metaphase was 19% for 15 min renaturation time and 16% for 30 min renaturation time. For the D15Z1 probe, generally four bright spots were visible and tentatively assigned according to chromosome length and centromere position (chromosomes 15 and 9). The intensity variation of each two homologues in the same metaphase spread showed a coefficient of variation of 47% (15 min) and 22% (30 min) for chromosome 15, and 19% (15 min) and 15% (30 min) for chromosome 9. The results indicate that the applied technique can considerably accelerate the FISH procedure and is suited for quantitative microscopy.

A simplified combination of DNA probe preparation and fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) has found widespread applications in cytogenetics. So far the standard protocols for probe amplification (and simultaneous labeling) by PCR, nick translation and in situ hybridization involve different buffer systems leading to a number of time consuming washing steps even before hybridization. In this manuscript we show a fast technique of a close combination of DNA probe preparation and in situ hybridization (ISH). This method was applied to metaphase chromosomes from human lymphocytes fixed on slides. Two specific repetitive DNA probes, the pUC 1.77 DNA probe and the DYZ1 repetitive DNA fraction were used, amplified and labeled in different ways. Additional experiments with total genomic male human DNA as the DNA probe suggest that this method may be extended to a large variety of other probes. Moreover the ISH technique described does not require toxic denaturing agents, such as formamide.

PCR amplification and simultaneous digoxigenin incorporation of long DNA probes for fluorescence in situ hybridization.

Nonradioactive in situ hybridization has found widespread applications in cytogenetics. Basic requirements are DNA probes in sufficient amounts and of high specificity as well as a labeling protocol of good reproducibility. The PCR has been of fundamental importance for the amplification of DNA sequences and thus for the production of DNA probes. Meanwhile, PCR protocols for amplification of DNA have reached a high degree of automation. So far, incorporation of labeled nucleotides into these DNA probes has normally been done by nick translation. Here we show that in using the PCR, amplification of a DNA probe larger than one kilobase accompanied by simultaneous incorporation of digoxigenin-11-dUTP can be performed for in situ hybridization experiments. As an example, the DNA probe pUC 1.77 specific for the subcentromeric region q12 of chromosome number 1 was used and hybridized against metaphase chromosomes from human lymphocytes. The labeled chromosome region was detected by anti-digoxigenin-fluorescein, Fab fragments. The experiments were evaluated by digital image analysis of microphotographs.