The spinal muscular atrophy disease gene product, SMN: A link between snRNP biogenesis and the Cajal (coiled) body.

The spliceosomal snRNAs U1, U2, U4, and U5 are synthesized in the nucleus, exported to the cytoplasm to assemble with Sm proteins, and reimported to the nucleus as ribonucleoprotein particles. Recently, two novel proteins involved in biogenesis of small nuclear ribonucleoproteins (snRNPs) were identified, the Spinal muscular atrophy disease gene product (SMN) and its associated protein SIP1. It was previously reported that in HeLa cells, SMN and SIP1 form discrete foci located next to Cajal (coiled) bodies, the so-called "gemini of coiled bodies" or "gems." An intriguing feature of gems is that they do not appear to contain snRNPs. Here we show that gems are present in a variable but small proportion of rapidly proliferating cells in culture. In the vast majority of cultured cells and in all primary neurons analyzed, SMN and SIP1 colocalize precisely with snRNPs in the Cajal body. The presence of SMN and SIP1 in Cajal bodies is confirmed by immunoelectron microscopy and by microinjection of antibodies that interfere with the integrity of the structure. The association of SMN with snRNPs and coilin persists during cell division, but at the end of mitosis there is a lag period between assembly of new Cajal bodies in the nucleus and detection of SMN in these structures, suggesting that SMN is targeted to preformed Cajal bodies. Finally, treatment of cells with leptomycin B (a drug that blocks export of U snRNAs to the cytoplasm and consequently import of new snRNPs into the nucleus) is shown to deplete snRNPs (but not SMN or SIP1) from the Cajal body. This suggests that snRNPs flow through the Cajal body during their biogenesis pathway.

Semithin cryosections as a tool to perform high resolution immunofluorescence and in situ hybridization analysis of the nervous tissue: a study in the supraoptic nucleus.

Immunofluorescence and fluorescence in in situ hybridization represent powerful approaches to correlate biochemical and molecular data with the structural organization of cells and tissues. However, the analysis of tissues by fluorescence microscopy is limited by the fact that most methods currently used to preserve the morphological integrity of sectioned samples at high resolution do not allow access of the labeled probes to the target molecules. Here we have made use of semithin cryosections obtained from rat supraoptic nucleus to perform immunofluorescence with antibodies directed against cytoplasmic and nuclear antigens, as well as fluorescence in situ hybridization with antisense oligonucleotide probes complementary to the poly(A) tail of mRNA and to specific mRNAs. In addition, DNA was visualized by incubation of sections with digoxigenin-labeled nucleotides in the presence of Escherichia coli DNA polymerase I. The high resolution of this DNA staining in combination with immunolabeling for nuclear antigens provides a powerful tool to analyze the structural and functional compartmentalization of neuronal cell nuclei. The major conclusion from this study is that performing fluorescence microscopy on 1 micron-thick cryosections provides an important tool to accurately localize proteins, DNA and RNA within nervous tissue in general and particularly in the model of supraoptic nucleus. Moreover, the cryosectioning technique appears particularly suited to the study of the localization of specific mRNA species in the neuronal cytoplasm and represents a useful approach to addressing the functional significance of mRNA localization in protein targeting.

The "accessory body" of Cajal in the neuronal nucleus. A light and electron microscopic approach.

The present light and electron microscopic study deals with the morphology and staining properties of two intranuclear inclusions - the "accessory body" of Cajal and the "coiled body"--in the supraoptic nuclei of adult rat hypothalamus, and supports the assumption that these structures represent the same intrinsic component of the neuronal nucleus. Consequently, we propose to term it "accessory body". The structure of this body was visualized by several different staining procedures: conventional electron microscopic techniques, a silver reaction, and the regressive EDTA staining for ribonucleoproteins. The silver-impregnation method employed here, which consists of a silver development sequence on hypothalamic tissue blocks prior to plastic embedding, permitted the study of supraoptic neurons at both light and electron microscopic levels. The nature and origin of "accessory bodies" are suggested and their possible functional role is briefly discussed.

Nuclear inclusions in immature glial cells of the rat hypothalamus during the postnatal period.

Ultrastructural study of the hypothalamic ventromedial nucleus during the postnatal period showed the presence of nuclear inclusions in immature glial cells. These inclusions, identified as "coiled bodies", consist of round-to-oval formations of coiled electron-dense strands embedded in a less dense fibrillar matrix. Coiled bodies are located free within the nucleoplasm, showing no specific relationships with the nucleolus or the nuclear membrane. The cells containing coiled bodies were typified as oligodendrocyte precursors, mainly oligodendroblasts. The coiled bodies were not found in mature glial cells nor in other types of immature glial elements. The nature and possible functional role of coiled bodies are suggested in the light of recent morphological and biochemical data.

Silver staining of the neuronal nucleus in electron microscopy: nucleolar organization in supraoptic nuclei of the rat hypothalamus.

The present paper describes a simple, efficient method for silver impregnation of supraoptic nuclei of the rat hypothalamus using a modification of the ammoniacal silver technique of Cajal (1903). This procedure, involving a silver-developer sequence in tissue blocks prior to plastic embedding, permits the simultaneous study of Ag-impregnated supraoptic neurons at both light and electron microscopic levels. Visualization of secretory magnocellular neurons impregnated by this technique using the electron microscope reveals a good preservation of nuclear structures. A selective accumulation of silver grains was observed over heterochromatin clumps and nucleoli, which allows the identification of the nucleolar fibrillar centers and also the dense fibrillar component as the main areas involved in the silver reaction. The meaning of such a silver-distribution pattern is discussed in the light of recent ultrastructural and biochemical data.

Coiled bodies in supraoptic nuclei of the rat hypothalamus during the postnatal period.

In electron microscopic studies of the supraoptic nuclei of the rat hypothalamus, structures identified as "coiled bodies" were found in magnocellular neurons. Although they could be seen elsewhere in mature neurosecretory cells, coiled bodies were commonly encountered in developing neurons during the postnatal period in both sexes. They appeared as distinctive nuclear inclusions consisting of round-to-oval networks of short electron-dense strands embedded in a less dense, fibrillar matrix, and lacking a limiting membrane. In fine structure and stain-affinity, they bore a resemblance to the fibrillar component of the nucleolus. Coiled bodies were located either in close association with the nucleolus or free within the nucleoplasm, showing no specific relationships with the perinucleolar chromatin or with the nuclear envelope. Their origin and functional meaning is discussed in the light of recent ultrastructural and biochemical data on cellular differentiation and nucleolar behavior.

Ciliated neurons in supraoptic nucleus of rat hypothalamus during neonatal period.

Cilia 9+0 have been found in differentiating neurosecretory neurons of the hypothalamic supraoptic nuclei of Wistar rats. These ciliated neurosecretory cells were always observed during the neonatal period of the rat and no more than one cilium per cell has been encountered. Single cilia arising from typical basal bodies were usually located in deep invaginations of the neuronal perikaryon, but can also be seen in superficial positions. Their basal bodies were commonly found in the vicinity of Golgi complexes, and associated structures such as striated rootlets and alar sheets were also present. In addition, single centrioles frequently appeared in these neurons, occurring close to basal bodies but also in centrosomatic areas. The respective roles of these morphological features are suggested and their possible ontogenetic significance is briefly discussed.

Subsurface cisterns in paraventricular nuclei of the hypothalamus of the rat.

Structures identified as subsurface cisterns (SSC's) were found in neurons of the paraventricular nuclei of the rat hypothalamus. They appeared as cytoplasmic organelles consisting most often of stacks of parallel cisterns apposed to the neuronal plasmalemma. These SSC's were located in the interneurons of the parvocellular system, but not in neurosecretory cells and glial cells. SSC's were seen at zones of cytoplasm apposed to neuronal or glial cell processes, showing in some instances specific relationships with synaptic areas. The morphological features of these SSC's are described, and their possible functional significance is briefly discussed.

Nuclear inclusions in paraventricular nucleus neurons of the rat hypothalamus.

This paper deals with the ultrastructure of two types of intranuclear inclusions, "microfilamentous spindle-shaped" and "crystalloid", present in paraventricular nucleus neurons of adult normal rats. These inclusions appear occasionally in some non-secretory neurons of the parvocellular system, but have never been seen in neurosecretory cells of the magnocellular system. The microfilamentous spindle-shaped inclusions show a close spatial relationship with the granulofibrillar body and interchromatin granules. The distribution and functional significance of such structures are discussed in the light of recent ultrastructural and biochemical studies on nuclear inclusions.

Intranuclear inclusions in pericytes of the hypothalamus of the rat.

This paper deals with the ultrastructure of two types of intranuclear inclusions, "nuclear bodies" and "membranous lamellar bodies", present in hypothalamic pericytes of intact adult rats. The nuclear bodies exhibited "simple" and "granular" forms, whereas the membranous lamellar bodies were entirely made up of myelin-like membrane whorls. The occurrence of these bodies in nuclei of pericytes has never been previously reported. The origin and functional meaning of such structures is discussed in the light of recent ultrastructural and biochemical studies on nuclear inclusions.

Ultrastructural study of pericytes in the rat supraoptic nucleus.

Pericytes of the supraoptic nucleus of normal rats have been studied with the electron microscope. These cells are morphologically similar to those of pericytes in other parts of the nervous system. The pericytes and their cytoplasmic processes were surrounded by basal membrane. The nucleus contained large masses of heterochromatin. The cytoplasm, less dense than that of the endothelial cells, contained numerous free ribosomes, cisternae of granular endoplasmic reticulum, various dictyosomes of the Golgi complex, isolated microtubules, a few mitochondria, and, occasionally, a diplosome. The presence of numerous lysosomes in some pericytes suggested that the cells are phagocytic even in normal circumstances.