Chemoreceptors expressed in taste, olfactory and male reproductive tissues.

We have identified three genes encoding previously uncharacterized chemoreceptors expressed in rat sensory and reproductive tissues using a reverse transcriptase polymerase chain reaction strategy. Degenerate oligonucleotides designed from conserved sequences in the rat olfactory receptor gene family were used to amplify candidate receptor gene products expressed in taste tissue. Sequence analysis of three distinct clonal isolates revealed that the gene products from taste bud were 30-75% identical to previously identified olfactory receptor genes. The genomic coding sequences predicted protein structures with seven membrane spanning regions that have strong conservation relative to other members of the G-protein-coupled olfactory receptor gene family. Transcripts for each of the three gene products were detected exclusively in taste, olfactory and male reproductive tissue. Sequence analysis of the polymerase chain reaction products confirmed that identical transcripts were expressed in all three tissues. These findings are the first demonstration that identical olfactory receptor-like gene are expressed in three distinct tissues.

Induction of L1 mRNA in PC12 cells by NGF is modulated by cell-cell contact and does not require the high-affinity NGF receptor.

We examined the effects of nerve growth factor (NGF) and cell-cell contact on expression of the neural cell adhesion molecule L1 in PC12 cells. After 7 d exposure to NGF, but not after exposure to EGF, FGF, TGF beta, or dibutyryl cAMP (dbcAMP), L1 mRNA levels increased fourfold. This increase was not blocked by K252a, an inhibitor of the high-affinity NGF receptor, although neurite extension was completely inhibited. L1 mRNA levels also increased in NGF-treated mutant PC12 cells (PC12nnr5) that lack the high-affinity NGF receptor. The effect of NGF on L1 mRNA was greatest in cells cultured at high density, but its effect on cells cultured at low density was augmented by antibody to L1 (to mimic L1 homophilic binding). Various extracellular matrix components had no differential effects on L1 mRNA levels in either the presence or absence of NGF. Together, these findings suggest that NGF regulates L1 expression by a mechanism that is independent of the high-affinity NGF receptor and that this regulation is modulated by cell-cell contact but not by cell-extracellular matrix interactions.

Expression of the neural cell adhesion molecule (NCAM) and polysialic acid during taste bud degeneration and regeneration.

Taste receptor cells are replaced throughout life, accompanied by continuing synaptogenesis between newly formed taste cells and first-order gustatory fibers. The neural cell adhesion molecule (NCAM) is expressed by a subset of taste cells in adult rodents and appears on gustatory nerve fibers during development prior to differentiation of the taste buds. We employed antibodies against the extracellular domain of the NCAM polypeptide (mAb 3F4) and against polysialic acid (PSA) residues found on embryonic forms of NCAM (mAb 5A5) to investigate the relationship between the expression of these molecules and the innervation of taste buds in adult rats. In unoperated rats, anti-NCAM recognized a subset of cells within the vallate taste buds and also the fibers of the glossopharyngeal (IXth) nerve, including those innervating the gustatory epithelium. Taste bud cells did not express PSA but mAb 5A5 immunoreactivity was observed on some fibers of the IXth nerve, including a few that entered the taste buds. Bilateral crush of the IXth nerve resulted in the loss of NCAM expression from the gustatory epithelium within 8 days. As IXth nerve fibers reinnervated the epithelium, NCAM expression was seen first in the nerve, followed by increased expression in the epithelium as the taste cells differentiated from their precursors. PSA expression by fibers of the IXth nerve did not return to normal until well after the regeneration of the vallate taste buds. The present results demonstrate that taste cell expression of NCAM is dependent upon innervation by the IXth nerve and that NCAM expression appears in the nerve prior to its expression in the differentiating epithelium during regeneration.(ABSTRACT TRUNCATED AT 250 WORDS)

VASE exon expression alters NCAM-mediated cell-cell interactions.

The neural cell adhesion molecule (NCAM) is found on cells as several related polypeptides formed by alternative splicing of the single NCAM gene. The alternatively spliced 30-bp VASE exon in the fourth immunoglobulin-like domain is the structural variation nearest those portions of the polypeptide proposed to mediate cell-cell adhesion. To test the ability of distinct forms of the NCAM molecules to mediate cell adhesion, L cells were transfected with expression vectors encoding rat 140 kD NCAM +/- the VASE exon. L cell lines which expressed these polypeptides were isolated and tested for self-aggregation in a low shear, rapid aggregation assay. Increased cellular aggregation of the transfectants was observed to be a function of the NCAM molecule expressed. These transfected cells showed segregation in a long term co-aggregation assay: cells expressing NCAM--VASE formed aggregates which tended to exclude cells expressing NCAM+VASE and vice versa. These results provide direct evidence that this small difference in NCAM structure is sufficient to allow segregation of cells.

Taste bud expression of human blood group antigens.

Some human blood group antigens are expressed by rodent epithelial cells at different stages of differentiation. Since adult taste cells are continually replaced throughout life, we investigated the expression of the H, B, A and Lewisb blood group determinants by cells of the rat fungiform, foliate and vallate papillae. We employed antibodies against the trisaccharide structures of the H, B, and A blood group antigens and against the Lewisb blood group epitope in studies of normal and denervated taste buds. The antibody against the H antigen reacted with the majority of cells in all taste buds and with cells in the spinous layer of the tongue epithelium. The B antigen was expressed by the majority of taste cells but not by other epithelial cells. The expression of the A antigen was significantly less in the fungiform taste buds than in the vallate or foliate taste buds. The A antigen was also abundantly expressed in the acini of the lingual salivary glands. The Lewisb epitope was expressed by a subset of cells in taste buds of the fungiform, foliate and vallate papillae. Taste buds are trophically dependent upon gustatory nerve innervation. Transection of the chorda tympani or the IXth nerve resulted in the loss of expression of these molecules from the gustatory epithelium, indicating that they are expressed only on differentiated taste cells. The blood group antigens are lactoseries carbohydrates; they are differentially expressed in developing cochlear hair cells and olfactory neurons and may play roles in cell-cell recognition, adhesion, and other interactions important in the developing nervous system. They could have similar functions in the taste and olfactory systems, where the receptors are continually renewed and new synapses between the receptors and their neural targets continually form.

NCAM expression by subsets of taste cells is dependent upon innervation.

The expression of the neural cell adhesion molecule (NCAM) and distinct carbohydrate groups by cells of the taste buds of the rat vallate papilla was investigated by immunohistochemical and biochemical techniques. We employed antibodies against 1) the extracellular (mAb 3F4) and cytoplasmic (mAb 5B8) portions of the NCAM polypeptide, 2) the highly sialylated form of NCAM (mAb 5A5), 3) carbohydrate epitopes associated with glycosylated NCAM forms in the rat (mAb 2B8) or frog (mAb 9-OE) olfactory system, and also 4) the Lewisb blood group carbohydrate epitope (mAb CO431). NCAM mRNA was demonstrated by polymerase chain reaction (PCR) in samples of the vallate papilla, suggesting the presence of NCAM in cells of the taste buds. Antibodies against NCAM (mAbs 3F4 and 5B8) recognized a subset (about 20%) of cells within the vallate taste buds; fibers of the glossopharyngeal nerve, including those innervating the gustatory epithelium, were NCAM immunoreactive. Taste bud cells did not express polysialic acid (mAb 5A5), but mAb 5A5 immunoreactivity was observed on fibers of the IXth nerve, including a few that entered the taste buds. All or nearly all of the cells within the vallate taste buds were immunoreactive to mAb 2B8, whereas mAbs 9-OE and CO431 reacted with subsets of cells. The carbohydrates recognized by mAbs 2B8 and 9-OE were also abundantly expressed in the ducts and acini of the lingual salivary glands. Bilateral crush of the IXth nerve resulted in the loss of expression of all of these molecules from the gustatory epithelium. If cells of the taste bud express NCAM during their final stage(s) of differentiation, then NCAM could play a role(s) in the growth of gustatory axons toward their target epithelial cells and in the recognition between the nerve fibers and mature taste receptor cells, or among the taste bud cells themselves.

Distinct subsets of sensory olfactory neurons in mouse: possible role in the formation of the mosaic olfactory projection.

The axons of the primary sensory olfactory neurons project from the olfactory neuroepithelium lining the nasal cavity, onto glomeruli covering the surface of the olfactory bulb. Neuroanatomical studies have shown previously that individual olfactory glomeruli are innervated by neurons that are dispersed widely within the nasal cavity. The aim of the present study was to test the hypothesis that phenotypically unique subsets of primary sensory olfactory neurons, scattered throughout the nasal cavity, project to a subset of glomeruli in specific olfactory bulb loci. Immunochemical and histochemical analyses in neonatal mice revealed that the plant lectin, Dolichos biflorus agglutinin, bound to a subset of mature primary sensory olfactory neurons which express the olfactory marker protein. This subset of neurons was principally located in the rostromedial and dorsal portions of the nasal cavity and projected specifically to a subset of glomeruli in the rostromedial and caudodorsal portions of the olfactory bulb. Analysis of Dolichos biflorus-reactive axons revealed that these axons coursed randomly, with no evidence of their selective fasciculation, within the olfactory nerve. It was only at the level of the rostral olfactory bulb that a significant reorganisation of their trajectory was observed. Within the outer fibre layer of the bulb, discrete bundles of lectin-reactive axons began to coalesce selectively into fascicles which preferentially oriented toward the medial side of the olfactory bulb. These data demonstrated that a phenotypically distinct subset of primary sensory olfactory neurons exhibits a topographical projection from the olfactory epithelium to the olfactory bulb, and suggests that these, and other subsets, may form the basis of the mosaic nature of this pathway. Moreover, it appears that the outer nerve fibre layer in the rostral olfactory bulb plays an important instructive role in the guidance and fasciculation of olfactory sensory axons.

A homopurine:homopyrimidine sequence derived from the rat neuronal cell adhesion molecule-encoding gene alters expression in transient transfections.

A 178-bp homopurine-homopyrimidine (R:Y) sequence is located upstream from the transcription start point (tsp) of the rat neuronal cell adhesion molecule-encoding gene (NCAM). This R:Y sequence contains several mirror repeats. Such sequences have been proposed to regulate gene expression. To determine its effect on gene transcription, a DNA fragment containing the R:Y sequence was cloned into a luciferase-encoding (luc) expression vector. Transient transfection assays with the R:Y-luc constructs were performed in cell lines which constitutively express (B104 rat neuronal cells and C6 rat glial cells) or lack (H411E rat liver cells and L mouse fibroblast cells) NCAMs. In its natural orientation, the R:Y sequence caused a 2.5-fold reduction in luc expression in B104 and H411E cells, but had a statistically insignificant effect in C6 and L cells. The magnitude of the R:Y sequence reduction in luc expression was position and orientation dependent (varying from 2- to 5.5-fold). To determine if nuclear protein(s) specifically bind the sequence, gel retardation assays of a DNA fragment containing the R:Y sequence were carried out with nuclear extracts from these four cell lines. Specific DNA-protein interaction was found with B104 and H411E nuclear extracts, but not with C6 and L cell nuclear extracts. Competition experiments indicate that the (AGG):(TCC) repeat segment within the rat R:Y sequence may constitute the protein-binding domain. These results indicate that the R:Y sequence may have a negative effect on gene transcription in certain cell lines. In correlation with this negative effect, these same cell lines also contain nuclear proteins which bind the sequence.

Axon growth is enhanced by NCAM lacking the VASE exon when expressed in either the growth substrate or the growing axon.

The neural cell adhesion molecule NCAM exists as several related peptides formed by alternative splicing of the single NCAM gene. Here the ability of NCAM containing and lacking the alternatively spliced VASE exon to act as a permissive growth substrate was tested by examining retinal axon outgrowth on normal L cell fibroblasts and L cells expressing stably transfected 140 kD NCAM +/- VASE. L cells expressing either NCAM form were a more permissive substrate than control L cells. At higher substrate cell densities, greater axon outgrowth occurred on substrate cells expressing NCAM - VASE than on those expressing NCAM + VASE. Similar experiments tested retinal axon growth on neuronal substrates by utilizing clonal B35 cells, C3 cells that are NCAM lacking variants of B35, and C3 cells into which 140 kD NCAM +/- VASE has been restored by transfection. Axon growth on C3 cells transfected with NCAM - VASE was greater than that on all other substrates including cells transfected with NCAM + VASE. In these experiments C3 cells and transfected C3 expressing NCAM + VASE cell promoted similar outgrowth. The influence on neurite growth of the NCAM isoform of the neurite itself was tested by examining neurite formation using combinations of C3 cells and C3 NCAM transfectants both in the growth monolayer and as responding cells. C3 cells were able to extend neurites, indicating NCAM is not required for neurite growth. However, C3 derivatives transfected with NCAM +/- VASE had greater neurite outgrowth. The most extensive neurite growth was found when NCAM - VASE was expressed by both substrate cells and the responding neurite growing cells. Thus NCAM enhances axon or neurite outgrowth when present either in the growth substrate or on the growing axon. NCAM - VASE has a significantly greater growth promoting capability than NCAM + VASE. The expression of NCAM + VASE by more mature neural cells could thus be a significant factor in the reduced axonation capabilities of mature neurons.

Olf-1-binding site: characterization of an olfactory neuron-specific promoter motif.

We report characterization of several domains within the 5' flanking region of the olfactory marker protein (OMP) gene that may participate in regulating transcription of this and other olfactory neuron-specific genes. Analysis by electrophoretic mobility shift assay and DNase I footprinting identifies two regions that contain a novel sequence motif. Interactions between this motif and nuclear proteins were detected only with nuclear protein extracts derived from olfactory neuroepithelium, and this activity is more abundant in olfactory epithelium enriched in immature neurons. We have designated a factor(s) involved in this binding as Olf-1. The Olf-1-binding motif consensus sequence was defined as TCCCC(A/T)NGGAG. Studies with transgenic mice indicate that a 0.3-kb fragment of the OMP gene containing one Olf-1 motif is sufficient for olfactory tissue-specific expression of the reporter gene. Some of the other identified sequence motifs also interact specifically with olfactory nuclear protein extracts. We propose that Olf-1 is a novel, olfactory neuron-specific trans-acting factor involved in the cell-specific expression of OMP.

Distinct NCAM splicing events are differentially regulated during rat brain development.

Primary transcripts for the neural cell adhesion molecule NCAM are highly alternatively spliced potentially giving rise to over 100 different mRNA forms. These mRNAs encode three major polypeptide isoforms of 120, 140, and 180 kDa each of which is thought to be composed of a mixture of polypeptides that differ by the variable presence of small exons at two locations. These NCAM 'microsplicing' patterns were examined within mRNA populations encoding each of the major isoforms to identify exactly which NCAM forms are present during brain development. The proportion of NCAM mRNAs containing at the exon 7/8 junction the alternatively microspliced 30 bp VASE exon increased similarly during brain development in mRNAs encoding all three major size classes. Perinatal brain, cultures of neurons from embryonic rats, and of glia from newborn rats all had low VASE levels while about 50% of the mRNAs in adult brain expressed VASE. In contrast, microsplicing at the exon 12/exon 13 junction was differentially regulated among NCAM major size classes. mRNAs containing microspliced exons totaling 3.15, or 18 base pairs (bp) represented greater than 50% of the total mRNAs encoding the 120 kDa forms at all ages. However, these exons were present in less than 15% of the 140 and 180 kDa encoding mRNAs in rats older than embryonic day 15. Similar results at the exon 12/13 junction were observed with mRNAs from neuronal cultures while glial cultures had greater levels of a 3 bp pair exon at this junction.(ABSTRACT TRUNCATED AT 250 WORDS)

From genotype to olfactory neuron phenotype: the role of the Olf-1-binding site.

The highly organized pattern of gene expression leading to the determination of cellular phenotype derives from the interplay between genetic and epigenetic factors. This is mediated in part by distinctive DNA sequence motifs present in the regulatory regions of various genes and the transcription factors with which they interact. The phenotype of olfactory neurons is determined in part by the selective expression of novel isoforms of several genes involved in chemosensory transduction. To characterize the mechanisms determining olfactory neuron phenotype we have been studying the olfactory marker protein (OMP), the first olfactory-specific protein to be isolated and cloned. The temporal and spatial expression of OMP is regulated stringently and is highly restricted to mature olfactory neurons in all vertebrates from amphibians to humans. Identification of the specific elements responsible for regulating the expression of the OMP gene will elucidate the mechanisms leading to the determination of olfactory neuron phenotype. Using a combined in vivo (transgenic mice) and in vitro (electrophoretic mobility shift assays and DNase I footprinting) approach, we have identified and characterized a novel genomic motif that binds an olfactory tissue nuclear protein(s) that we designate Olf-1. We propose that Olf-1 is a novel olfactory-specific transacting factor responsible for directing the expression of genes containing the Olf-1 motif in olfactory neurons. Thus it may play a role in regulating the expression of genes associated with neuronal turnover and olfactory transduction.

The neural cell adhesion molecule (NCAM) heparin binding domain binds to cell surface heparan sulfate proteoglycans.

The neural cell adhesion molecule (NCAM) has been strongly implicated in several aspects of neural development. NCAM mediated adhesion has been proposed to involve a homophilic interaction between NCAMs on adjacent cells. The heparin binding domain (HBD) is an amino acid sequence within NCAM and has been shown to be involved in NCAM mediated adhesion but the relationship of this domain to NCAM segments mediating homophilic adhesion has not been defined. In the present study, a synthetic peptide corresponding to the HBD has been used as a substrate to determine its role in NCAM mediated adhesion. A neural cell line expressing NCAM (B35) and its derived clone which does not express NCAM (B35 clone 3) adhered similarly to plates coated with HBD peptide. A polyclonal antiserum to NCAM inhibited B35 cell-HBD peptide adhesion by only 10%, a value not statistically different from inhibition caused by preimmune serum. Both these experiments suggested no direct NCAM-HBD interactions. To test whether the HBD peptide bound to cell surface heparan sulfate proteoglycans (HSPG), HSPG synthesis was inhibited using beta-D-xyloside. After treatment, B35 cell adhesion to the HBD peptide, but not to control substrates, was significantly decreased. B35 cell adhesion to the HBD peptide could be inhibited by 10(-7) M heparin but not chondroitin sulfate. Preincubation of the substrate (HBD peptide) with heparin caused dramatic reduction of B35 cell-HBD peptide adhesion whereas preincubation of B35 cells with heparin caused only modest reductions in cell-HBD adhesion. Furthermore, inhibition of HSPG sulfation with sodium chlorate also decreased the adhesion of B35 cells to the HBD peptide. These results strongly suggest that, within the assay system, the NCAM HBD does not participate in homophilic interactions but binds to cell surface heparan sulfate proteoglycan. This interaction potentially represents an important mechanism of NCAM adhesion and further supports the view that NCAM has multiple structurally independent binding sites.

Lens structures exist transiently in development of transgenic mice carrying an alpha-crystallin-diphtheria toxin hybrid gene.

The development of lens structures in transgenic mice (lnl mice) which carry the diphtheria toxin A chain-coding sequence under the control of the alpha-crystallin promoter is examined here in detail. The initial stages of lens development during embryonic days 10.5 to 12.5 (E10.5-E12.5), including the invagination of the surface ectoderm to form a lens vesicle, closure of the vesicle to form a lens cup, and initial appearance of the lens itself, appeared identical in histologic analyses of lnl mice and genotypically wild-type littermate controls. However, by E12.5, cells in the central posterior lens of developing lnl mice appeared to be vacuolated and undergoing necrosis. This necrosis was quite prominent at E14.5 and the overall lens size was significantly reduced. The lenses of lnl mice continued to be present but were significantly smaller throughout embryonic development. The cells of these lenses were capable of undergoing biochemical differentiation, reacting with antibodies to both alpha- and beta-/gamma-crystallin. alpha-Crystallin expression was initiated at the appropriate time (E10.5) and maintained in most cells of lnl lenses. The expression of beta-/gamma-crystallins was surprising as these crystallins are expressed later in lens development after normal expression of alpha-crystallin and after the anticipated time of expression of the diphtheria toxin transgene. Despite extensive necrosis and cell death, lens structures persisted in lnl mice and disappeared only in the early postnatal period between days 3 and 6. Throughout the perinatal period, the remaining lens cells expressed both alpha- and beta-/gamma-crystallins. Prenatal development of the retina and ciliary body was relatively normal although the eye was significantly reduced in overall size. Some additional developmental defects were noted including persistent hyaloid artery and thickened cornea. In the perinatal period the rapidly expanding retina filled the entire eye leaving essentially no anterior or posterior chamber. These results clearly indicate that lens cells which are the target of diphtheria toxin-mediated cell ablation techniques persist for a significant time during development and thus place limitations on the interpretations of results obtained using this technique.

Agonist-activated cobalt uptake identifies divalent cation-permeable kainate receptors on neurons and glial cells.

Activation of kainate receptors causes Co2+ influx into neurons, type-2 astrocytes, and O-2A progenitor cells. Agonist-activated Co2+ uptake can be performed using cultured cells or fresh tissue slices. Based on the pattern of response to kainate, glutamate, and quisqualate, three functionally different kainate-activated ion channels (K1, K2, and K3) can be discriminated. Co2+ uptake through the K1 receptor was only activated by kainate. Both kainate and glutamate activated Co2+ uptake through the K2 receptor. Co2+ uptake through the K3 receptor was activated by all three ligands: kainate, glutamate, and quisqualate. Co2+ uptake occurred through a nonselective cation entry pathway permeable to Co2+, Ca2+, and Mn2+. The agonist-dependent activation of divalent cation influx through different kainate receptors could be correlated with expression of certain kainate receptor subunit combinations. These results are indicative of kainate receptors that may contribute to excitatory amino acid-mediated neurotoxicity.

At least 27 alternatively spliced forms of the neural cell adhesion molecule mRNA are expressed during rat heart development.

The major membrane-associated or transmembrane isoforms of the neural cell adhesion molecule (NCAM) are generated by alternative splicing at the 3' end of the mRNA. Further diversity in NCAM structure is observed in the extracellular region of the polypeptide, where the insertion of additional amino acid residues can result from alternative splicing events occurring at the exon 7-exon 8 and exon 12-exon 13 junctions. Here we report the characterization of tissue-specific patterns of alternative splicing at the exon 12-exon 13 junction by using the polymerase chain reaction. Nine alternatively spliced sequences in rat heart between exon 12 and exon 13 were identified. Each sequence consisted of different combinations of the three small exons (15, 48, and 42 bp in length) and the AAG triplet that make up MSD1, the 108-bp muscle-specific sequence found in human skeletal muscle NCAM (G. Dickson, H.J. Gower, C. H. Barton, H. M. Prentice, V. L. Elsom, S. E. Moore, R. D. Cox, C. Quinn, W. Putt, and F. S. Walsh, Cell 50:1119-1130, 1987). Although the rat equivalent of MSD1 (designated 15+ 48+ 42+ 3+) was detected in all ages of heart examined, it was only one of four or five major splice combinations at any given age. The only alternatively spliced sequence found in the exon 7-exon 8 junction of heart NCAM mRNA was the 30-bp variable alternatively spliced exon previously identified in rat brain. Twenty-seven NCAM forms with distinct sequences were found by analysis of individual NCAM transcripts from postnatal day 1 heart tissue for alternative splicing at the exon 7-exon 8 junction, the exon 12-exon 13 junction and the 3' end. Several combinations of splicing patterns in these three different regions of the gene appeared to be preferentially expressed. The observation that the expression of alternatively spliced forms of NCAM is developmentally regulated suggests a role for NCAM diversity in cardiac development.

Delineation of olfactory pathways in the frog nervous system by unique glycoconjugates and N-CAM glycoforms.

The olfactory neuroepithelium, which contains the primary sensory olfactory neurons, continually undergoes neurogenesis and axonal outgrowth throughout life. We describe here several new olfactory system-specific glycoforms of the neural cell adhesion molecule N-CAM in the frog, R. catesbeiana. Using immunochemical methods for in situ localization, we show that the lectin dolichos biflorus agglutinin (DBA) and two monoclonal antibodies, 9OE and 3A6, detect three unique N-CAM forms present on primary sensory olfactory axons. In addition, DBA and monoclonal antibody 9OE recognize glycoconjugates and/or N-CAM glycoforms expressed specifically in discrete central olfactory pathways and regions in frog brain. This is a novel example of unique adhesion molecule forms present in a chain of two neurons within a vertebrate neural pathway. Together these glycoconjugates and N-CAM glycoforms may participate in cellular interactions associated with olfactory system pathway formation and renewal.

Immunochemical markers for the frog olfactory neuroepithelium.

Monoclonal antibodies (mAbs) were generated that react with the major cell types in the olfactory neuroepithelium of the frog, Rana catesbeiana. This pseudostratified epithelium consists of apical supporting cells, a middle layer of olfactory receptor neurons and a heterogeneous population of basal cells consisting of basal cells proper and globose basal cells. Both olfactory receptor neurons and globose basal cells were labelled by mAb 13-OE, which recognized the neural cell adhesion molecule NCAM. The identity of these NCAM positive cells was established by analysing regenerating olfactory epithelium and by a double-antibody labelling immunofluorescence technique. The olfactory nerve was lesioned, which induced the death of olfactory receptor neurons and the subsequent proliferation of basal cells. When the regenerating olfactory epithelium was analysed prior to the reconstitution of mature olfactory neurons, mAb 13-OE reacted specifically with globose basal cells and not the basal cells proper. Simultaneous labelling of normal olfactory epithelium with mAb 13-OE and polyclonal anti-keratin antibodies, the latter of which labels supporting cells and basal cells proper, revealed no double-labelled cells. These results further confirmed that NCAM was expressed by both globose basal cells and receptor neurons but not by other cell types within the epithelium. Additional cell types in the olfactory epithelium reacted with other new mAbs: 4-OE, 5-OE, 7-OE and 9-OE. Supporting cells were stained by mAb 4-OE. Olfactory receptor neurons and the entire population of basal cells were immunoreactive with mAb 7-OE. The cilia and knobs of receptor neurons were strongly immunoreactive with mAb 5-OE whereas mAb 9-OE selectively stained olfactory knobs and not the cilia on these chemosensory cells. These studies are a first step towards experimental approaches designed to elucidate the mechanisms underlying the unique proliferative properties of the olfactory neuroepithelium in frog.

Expression of the unique NCAM VASE exon is independently regulated in distinct tissues during development.

During development of the rat central nervous system, neural cell adhesion molecule (NCAM) mRNAs containing in the extracellular domain a 30-bp alternative exon, here named VASE, replace RNAs that lack this exon. The presence of this alternative exon between previously described exons 7 and 8 changes the predicted loop structure of the derived polypeptide from one resembling an immunoglobulin constant region domain to one resembling an immunoglobulin variable domain. This change could have significant effects on NCAM polypeptide function and cell-cell interaction. In this report we test multiple rat tissues for the presence of additional alternative exons at this position and also examine the regulation of splicing of the previously described exon. To sensitively examine alternative splicing, polymerase chain reactions (PCRs) with primers flanking the exon 7/exon 8 alternative splicing site were performed. Four categories of RNA samples were tested for new exons: whole brain from embryonic day 11 to adult, specific brain regions dissected from adult brain, clonal lines of neural cells in vitro, and muscle cells and tissues cultured in vitro and obtained by dissection. Within the limits of the PCR methodology, no evidence for any alternative exon other than the previously identified VASE was obtained. The regulation of expression of this exon was found to be complex and tissue specific. Expression of the 30-bp exon in the heart and nervous system was found to be regulated independently; a significant proportion of embryonic day 15 heart NCAM mRNAs contain VASE while only a very small amount of day 15 nervous system mRNAs contain VASE. Some adult central nervous system regions, notably the olfactory bulb and the peripheral nervous system structures adrenal gland and dorsal root ganglia, express NCAM which contains very little VASE. VASE is undetectable in NCAM PCR products from the olfactory epithelium. Other nervous system regions express significant quantities of NCAM both with and without VASE. Clonal cell lines in culture generally expressed very little VASE. These results indicate that a single alternative exon, VASE, is found in NCAM immunoglobulin-like loop 4 and that distinct tissues and nervous system regions regulate expression of VASE independently both during development and in adult animals.

Transcription initiation sites and structural organization of the extreme 5' region of the rat neural cell adhesion molecule gene.

Through analysis of rat genomic cosmid clones, the 5'-most exon of the rat neural cell adhesion molecule (NCAM) gene was identified. This exon, here named exon 0, contained the entire 5' untranslated region and the N-terminal signal sequence of the polypeptide. Exon 0 was isolated from a 1.6-kilobase (kb) EcoRI-HindIII fragment of rat genomic cosmid clone 9 which was 35 kb in length. This fragment was sequenced and found to contain approximately 940 base pairs (bp) of 5'-flanking sequence, exon 0, which was approximately 245 bp in length, and approximately 400 bp of the following intron 0. By using information derived from this fragment and the pR18 rat NCAM cDNA, the transcription initiation sites were determined with two assays. Both primer extensions and nuclease S1 protection assays of postnatal day 7 rat brain RNA identified seven initiation sites within a single 10-bp region at positions -195 to -186 relative to the translation start site. An additional minor site was found at position -329. In the immediate 5' region, no consensus TATA or CCAAT sequences were found. Potential regulatory elements within this region include Sp1 consensus binding sites and also a 178-bp homopurine-homopyrimidine sequence containing several mirror repeats. NCAM has multiple transcripts which are regulated in a developmental and tissue-specific fashion. To determine whether these transcripts are initiated at the same sites, transcription initiation sites were analyzed in postnatal day 7 and adult rat brain and also in cultured cell lines of neuronal, glial, and muscle phenotypes. These tissues and cells exhibited distinct NCAM transcript populations in Northern (RNA) dot blot analysis. In all cases similar transcription start sites were found, suggesting that all major NCAM transcripts have similar or identical initiation sites. These results provide essential information to begin analysis of NCAM regulation in different tissues and during development.