Detection of mRNA molecules coding for neuropeptide hormones of the pond snail Lymnaea stagnalis by radioactive and non-radioactive in situ hybridization: a model study for mRNA detection

To develop and optimize non-radioactive in situ hybridization techniques for mRNA detection, we used the neuropeptidergic system of the pond snail Lymnaea stagnalis as a biological model system. First, we investigated the in situ hybridization procedure using radioactive-labeled cDNA and synthetic oligonucleotide probes specific for egg-laying hormone (ELH) mRNA and molluscan insulin-like peptide (MIP) mRNA. The results show an intense grain deposit above the caudodorsal cells and light-green cells expressing, respectively, ELH mRNA and MIP mRNA. Good results with relation to signal strength and tissue morphology were obtained with freeze-dry paraformaldehyde vapor fixation. The necessity to perform tissue pre-treatment appeared to be dependent on the cell type of interest. The optimized in situ hybridization protocol proved to be applicable using probes that are either sulfonated/transaminated or labeled with acetylaminofluorene (AAF). In situ hybridization of such haptenized probes led to intense and specific staining of the cytoplasm of the caudodorsal cells. Egg-laying hormone mRNA appeared not to be homogeneously distributed in the cytoplasm but showed a "patch-like" pattern. Nuclear and axoplasmic staining for mRNA was also observed.     

Bi-color detection of two target DNAs by non-radioactive in situ hybridization

Summary A non-radioactive in situ hybridization technique is described which allows the simultaneous detection of different DNA sequences. To demonstrate the feasibility of the proccdure, metaphases and interphase nuclei of a human-mouse somatic cell hybrid were simultaneously hybridized with mercurated total human DNA and a biotinylated mouse satellite DNA probe. After the hybridization, the probes were detected immunocytochemically using two different and independent affinity systems. By this approach we visualized the two DNA target sequences in metaphase chromosomes and in interphase nuclei with FITC and TRITC fluorescence, or blue (alkaline phosphatase) and brown (peroxidase) precipitated enzyme products. This method not only allows detection of intact chromosomes but also the visualization of rearrangements between parts of human and mouse chromosomes. Furthermore, the technique demonstrates the high topological resolution of nonradioactive in situ hybridizations.This investigation was supported in part by FUNGO, Foundation of Medical Scientific Research in The Netherlands (grant nr 13-54-21)     

Assessment of decalcifying protocols for detection of specific RNA by non-radioactive in situ hybridization in calcified tissues

For the best performance of in situ analysis of specific RNA expression in calcified tissues, it is necessary to choose an appropriate protocol to decalcify the tissues. We evaluated the usefulness of various acid-based decalcifying reagents with reference to 28 S rRNA staining by in situ hybridization using a thymine-thymine dimerized oligonucleotide probe. The reagents evaluated were 10% nitric acid, 10% HCl, 5% formic acid, 5% trichloroacetic acid, Morse’s solution, Plank-Rychlo’s solution, and K-CX solution, all of which are commonly used to decalcify tissues, and their effects on retention of morphology and RNA were compared with EDTA-based solutions. When normal mouse mandible was used as a model tissue, well-preserved morphology of ameloblasts was obtained from sections decalcified with Morse’s solution, 10% HCl, Plank-Rychlo’s solution, and K-CX solution, and best retention of 28 S rRNA was obtained with 5% formic acid and Morse’s solution. We recommend Morse’s solution to decalcify tissues to be processed for the rapid analysis of specific RNA expression. Indeed, we detected specific mRNAs strongly in sections treated with Morse’s solution, and quantitative analysis showed that the ratio of signal intensities of 28 S rRNA and the specific mRNAs correlated with each other depending on decalcifying solutions. Accepted: 3 January 2000     

Peptidomics analysis of neuropeptides involved in copulatory behavior of the mollusk Lymnaea stagnalis

Male copulation behavior in mollusks is controlled by an array of peptide messengers. In the present study, we have used a peptidomics approach employing liquid chromatography in conjunction with electrospray mass spectrometry to characterize peptides contained in the penial complex of the freshwater snail, Lymnaea stagnalis. In addition to the previously described peptides, we have identified a group of novel peptides that share the carboxyl termini of -FVRIamide. A cDNA cloning study revealed the organization of the precursor, which contains 20 peptide domains with the carboxyl termini of -F(X)RIamide which are flanked by many putative proteolytic sites including the KR and the less commonly occurring (G)K and (G)R sites. In addition, there are several monobasic R and dibasic RR and KK sites that may be used for processing. We then used MALDI-TOF/TOF-MS in a data-dependent mode, which selected all the molecular ion species with the predicted masses of the mature -F(X)RIamide peptides, and performed MS/MS analysis on these peptides. This approach allowed us to identify all the predicted -F(X)RIamide peptides. Immunocytochemistry showed the localization of -FVRIamide immunoreactive neurons in several central ganglia, and immunoreactive axons in the penial complex. Finally, application of synthetic -FVRIamide peptides to an in vitro posterior vas deferens preparation showed inhibitory effect on the spontaneous contraction/relaxation cycle of the vas deferens.     

Use of whole cosmid cloned genomic sequences for chromosomal localization by non-radioactive in situ hybridization

Summary We report a general procedure which allows the application of whole cosmid cloned genomic sequences for non-radioactive in situ hybridization. The presence of highly repetitive sequences, like Alu and Kpn fragments, is eliminated through competition hybridization with Cot-1 DNA. The method has been tested and optimized with several randomly chosen cosmids of the human thyroglobulin (Tg) gene (8q24). At present, the procedure can be performed with three of the four tested individual cosmids. In cases where a single clone does not result in a specific signal, a larger fragment may be required, which can be accomplished by using two (partially overlapping) cosmids of the same region. The advantages and further potentialities of such a hybridization approach are discussed.     

The effect of hydroxyurea on ciliogenesis in ciliary epithelium of the mollusk Lymnaea stagnalis

Electron-microscopy study of the ciliary epithelium structure of the mollusk Lymnaea stagnalis was carried out under the action of hydroxyurea. By the method of radioautography, a high proliferative activity of the ciliary epithelium was established as the norm; a cluster distribution of cells, including the label, was noted. The presence of hydroxyurea in the mollusk organism was shown to inhibit proliferation. Scanning electron microscopy of the molluskan foot surface revealed clusters of nonciliated cells and of cells with short villi in control epithelial folds. Under hydroxyurea treatment for 24 h, such sites disappeared completely and ciliary epithelium looked uniform and was composed of cells with long cilia. By transmission electron microscopy, it was established that hydroxyurea did not affect the formation of the basal body and course of ciliogenesis. It has been suggested that hydroxyurea not only inhibits proliferative activity of epithelial cells, but also induces differentiation of unciliated into the ciliated cells.     

Detection of biogenic monoamines in the developing nervous system of Lymnaea stagnalis mollusk embryos]

Formaldehyde-induced fluorescence of intraneuronal monoamines can be demonstrated in the Lymnaea embryos from the "late veliger" stage on. Green specific fluorescence indicating the presence of a primary catecholamine occurs in two paired formations which contain a mass of fibres and varicosities. The formations are supposed to correspond to cerebral and pedal ganglia. Single fibres of the same type can be seen in the foot and other organs of the embryo.     

Detection of gastrin and its messenger RNA in Zollinger-Ellison tumors by non-radioactive in situ hybridization and immunocytochemistry.

Gastrin immunocytochemistry and non-radioactive in situ hybridization, using biotinylated oligonucleotide probes, for gastrin mRNA have been used for studying a retrospective material of six gastrin-producing (Zollinger-Ellison) tumors. Hybridization results for gastrin mRNA were positive in all six, while gastrin immunoreactivity could be detected in five tumors. In one of the patients, different areas of the same tumor displayed differences in immunoreactivity to gastrin, but were uniformly hybridization positive. Weak hybridization signals were detected in liver metastases from a necropsy case, while the gastrin immunostaining was more pronounced. The results show that non-radioactive hybridization methods are applicable to routine clinical specimens stored for as long as 16 years and that in situ hybridization may be a useful complement to immunocytochemical diagnosis, particularly in cases where high synthesis and little storage of hormonal products occur.     

Simultaneous detection of neuropeptides and messenger RNA in the magnocellular hypothalamo-neurohypophysial system by a combination of non-radioactive in situ hybridization histochemistry and immunohistochemistry

A protocol was developed combining non-radioactive in situ hybridization histochemistry with enzyme based immunohistochemistry, detect the expression of mRNA in phenotypically defined neurons. Freefloating brain sections were hybridized with the oligonucleotide probes which have been 3-end labelled with biotin-11-dUTP. The hybridized probe was visualized by a combined avidin-biotin bridge method, anti-avidin immunohistochemistry, and horseradish peroxidase detection using diaminobenzidine as a substrate. The in situ hybridization step yielded a very stable reaction product enabling subsequent immunohistochemical reactions using horseradish peroxidase and benzidine dihydrochloride as a chromogen. Magnocellular neurons of the hypothalamo-neurophypophysial system synthesize either vasopressin or oxytocin; water deprivation and chronic saline ingestion are potent stimuli for the expression of both of the genes encoding these neuropeptides. A number of other neuropeptides with putative transmitter action are synthesized in magnocellular neurons during such stimulation. Experiments were performed to explore whether neuropeptide Y immunoreactivity is present within magnocellular vasopressin mRNA-expressing neurons of the hypothalamo-neurophypophysial system. The results clearly demonstrated that neuropeptide Y-immunoreactive elements were present within a number of magnocellular vasopressin mRNA-containing cells. In addition, immunohistochemical detection of the neuropeptides ocytocin and cholecystokinin was carried out on sections hybridized non-radioactively for vasopressin; as expected vasopressin mRNA did not co-exist with cholecystokinin, whereas a few oxytocin immunoreactive neurons in osmotically stimulated animals also contained vasopressin mRNA. The developed method makes possible the immunohistochemical detection of intracellular antigens with concomitant detection of intracellular mRNA.     

Detection of gastrin and its messenger RNA in Zollinger-Ellison tumors by non-radioactive in situ hybridization and immunocytochemistry

Summary Gastrin immunocytochemistry and non-radioactive in situ hybridization, using biotinylated oligonucleotide probes, for gastrin mRNA have been used for studying a retrospective material of six gastrin-producing (Zollinger-Ellison) tumors. Hybridization results for gastrin mRNA were positive in all six, while gastrin immunoreactivity could be detected in five tumors. In one of the patients, different areas of the same tumor displayed differences in immunoreactivity to gastrin, but were uniformly hybridization positive. Weak hybridization signals were detected in liver metastases from a necropsy case, while the gastrin immunostaining was more pronounced. The results show that non-radioactive hybridization methods are applicable to routine clinical specimens stored for as long as 16 years and that in situ hybridization may be a useful complement to immunocytochemical diagnosis, particularly in cases where high synthesis and little storage of hormonal products occur.     

Localization of ovulation hormone-like neuropeptide in the central nervous system of the snail Lymnaea stagnalis by means of immunocytochemistry and in situ hybridization

Summary The caudo-dorsal cells (CDC) in the cerebral ganglia of the pond snail Lymnaea stagnalis synthesize the 36-amino acid ovulation hormone (CDCH). We have used immuno-cytochemistry and in situ hybridization to reveal the localization of neurons and axons containing CDCH-like material.A monoclonal antibody to a fragment of CDCH and a cDNA probe encoding CDCH reacted with the CDC-system, with specific cell groups in the cerebral and pleural ganglia, and with individually occurring neurons throughout the central nervous system. The cells in the pleural ganglia, which were found in about 50% of the preparations studied, are considered as ectopic CDC. They are morphologically similar to CDC in their somal dimensions and axonal organization. By means of immuno-electron microscopy it was shown that these neurons contain secretory vesicles that are similar to those of the CDC. The neurons of the bilateral groups occurring in the cerebral ganglia in addition to the CDC are smaller and more intensely stained than the CDC. Axons of these small neurons probably have varicosities located on the CDC axons in the neuropil of the cerebral ganglion, indicating synaptic contacts. Two major axon tracts could be followed from (or toward) the neuropil of the cerebral ganglion. One tract runs from the cerebral gangion via the pleural and parietal ganglia to the visceral ganglion, giving off branches to most nerves emanating from these ganglia. The other tract could be traced through the cerebro-pedal connective to the pedal ganglia. Only in the right pedal ganglion was extensive axonal branching observed. The nerves emanating from this ganglion contained many more immunoreactive axons than those from the left pedal ganglion. A polyclonal antibody raised against the synthetic fragment of CDCH stained, in addition to the neurons and axons revealed with the monoclonal antibody and the cDNA probe, three other major groups of neurons. Two are located in the cerebral ganglion, the other in the left pedal ganglion.The present findings suggest the presence of a system of neurons that contain CDCH or CDCH-like peptides. The role this system may play in the control of egg-laying and egg-laying behaviour is discussed.     

Electron microscopic investigation of a giant neuron identified in the right parietal ganglion of Lymnaea stagnalis (L)

The ultrastructure of a giant neuron (GRP1) identified in the right parietal ganglion of Lymnaea stagnalis was examined. The results suggest that GRP1 is a neurosecretory cell. The perikaryon is characterized by many neurosecretory-like granules of variable electron density. The average diameter (longer axis) of the granules was 1850 A. Furthermore highly-developed rough endoplasmic reticulum and Golgi complex were observed in the cytoplasm. Synapse-like structures were often found on the somatic membrane, but typical, true synaptic endings were not seen.     

Seasonal variation in the content of free reducing sugars in body fluids of freshwater mollusk Lymnaea stagnalis

Accumulation of certain mono-and disaccharides in body fluids of ectotherms in autumn and winter suggests their protective role in adaptation to near-zero temperatures. In this context, the effect of seasonal temperature variation on the content of free reducing sugars was studied in freshwater snail Lymnaea stagnalis. The summer and autumn carbohydrate spectrum essentially featured high concentrations of fructose and glucose. After 2.5-month hypobiosis (4°C), the concentration of fructose and glucose decreased 35 and 12 times, respectively. Maltose and other disaccharides (presumably cellobiose) undetectable in summer became detectable in autumn before hypobiosis started. The subsequent level of maltose increased to almost reach the sharply decreased glucose level and to exceed the fructose level after 2.5-month hypobiosis. The involvement of maltose in the mechanisms of L. stagnalis adaptation to near-zero temperatures as a membrane protectant is proposed.     

Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry.

In the present study some experimental parameters for in situ hybridization histochemistry (ISHH) have been analysed using 35S-labelled and alkaline phosphatase-conjugated probes, in order to develop a reproducible double-labelling procedure. We have compared the total exclusion of tissue fixation with tissue sections fixed by immersion in formalin. In addition, the effect of dithiothreitol was assessed both when combining radiolabelled and non-radioactive probes on a single tissue section and when the probes were used separately. Hybridization of unfixed tissue resulted in stronger specific labelling and lower background both for radiolabelled and alkaline phosphatase-conjugated probes. No loss in tissue preservation was seen at the light microscopic level after hybridization of unfixed tissue. High concentrations (200 mM) of dithiothreitol strongly suppressed background when using 35S-labelled probes, whereas in the non-radioactive procedure, alkaline phosphatase labelling could only be achieved with very low dithiothreitol concentrations (less than 1 mM). This incompatibility led to a protocol using unfixed tissue sections and a sequential hybridization procedure, with the radiolabelled probe and high concentrations of dithiothreitol in the first step and the alkaline phosphatase-conjugated probe without dithiothreitol in the second step.     

Double in situ hybridization for detection of Herpes simplex virus and cytomegalovirus DNA using non-radioactive probes.

We describe a double in situ hybridization assay for the simultaneous detection of Herpes simplex virus (HSV) and cytomegalovirus (CMV) DNA in infected cell cultures using non-radioactive-labeled probes. This work used a biotinylated HSV DNA probe, which can be revealed by an avidin-biotin-peroxidase complex and a digoxigenin-labeled CMV DNA probe, visualized by anti-digoxigenin F(ab) fragments conjugated with alkaline phosphatase. Light microscopy visualization was achieved by the contrasting colors of appropriate peroxidase and alkaline phosphatase reaction products (red and dark blue, respectively). The time required to perform the double hybridization assay was about 3 hr. This double hybridization assay proved to be sensitive, specific, and provided good resolving power.