Sorting of tropomyosin isoforms in synchronised NIH 3T3 fibroblasts: evidence for distinct microfilament populations.

The nonmuscle actin cytoskeleton consists of multiple networks of actin microfilaments. Many of these filament systems are bound by the actin-binding protein tropomyosin (Tm). We investigated whether Tm isoforms could be cell cycle regulated during G0 and G1 phases of the cell cycle in synchronised NIH 3T3 fibroblasts. Using Tm isoform-specific antibodies, we investigated protein expression levels of specific Tms in G0 and G1 phases and whether co-expressed isoforms could be sorted into different compartments. Protein levels of Tms 1, 2, 5a, 6, from the alpha Tm(fast) and beta-Tm genes increased approximately 2-fold during mid-late G1. Tm 3 levels did not change appreciably during G1 progression. In contrast, Tm 5NM gene isoform levels (Tm 5NM-1-11) increased 2-fold at 5 h into G1 and this increase was maintained for the following 3 h. However, Tm 5NM-1 and -2 levels decreased by a factor of three during this time. Comparison of the staining of the antibodies CG3 (detects all Tm 5NM gene products), WS5/9d (detects only two Tms from the Tm 5NM gene, Tm 5NM-1 and -2) and alpha(f)9d (detects specific Tms from the alpha Tm(fast) and beta-Tm genes) antibodies revealed 3 spatially distinct microfilament systems. Tm isoforms detected by alpha(f)9d were dramatically sorted from isoforms from the Tm 5NM gene detected by CG3. Tm 5NM-1 and Tm 5NM-2 were not incorporated into stress fibres, unlike other Tm 5NM isoforms, and marked a discrete, punctate, and highly polarised compartment in NIH 3T3 fibroblasts. All microfilament systems, excluding that detected by the WS5/9d antibody, were observed to coalign into parallel stress fibres at 8 h into G1. However, Tms detected by the CG3 and alpha(f)9d antibodies were incorporated into filaments at different times indicating distinct temporal control mechanisms. Microfilaments in NIH 3T3 cells containing Tm 5NM isoforms were more resistant to cytochalasin D-mediated actin depolymerisation than filaments containing isoforms from the alpha Tm(fast) and beta-Tm genes. This suggests that Tm 5NM isoforms may be in different microfilaments to alpha Tm(fast) and beta-Tm isoforms even when present in the same stress fibre. Staining of primary mouse fibroblasts showed identical Tm sorting patterns to those seen in cultured NIH 3T3 cells. Furthermore, we demonstrate that sorting of Tms is not restricted to cultured cells and can be observed in human columnar epithelial cells in vivo. We conclude that the expression and localisation of Tm isoforms are differentially regulated in G0 and G1 phase of the cell cycle. Tms mark multiple microfilament compartments with restricted tropomyosin composition. The creation of distinct microfilament compartments by differential sorting of Tm isoforms is observable in primary fibroblasts, cultured 3T3 cells and epithelial cells in vivo.

CROC-4: a novel brain specific transcriptional activator of c-fos expressed from proliferation through to maturation of multiple neuronal cell types.

A novel, brain-specific cDNA, denoted CROC-4, was cloned from human brain by a contingent replication of cDNA procedure capable of detecting transcriptional activators of the human c-fos proto-oncogene promoter. CROC-4 encoded an 18-kDa serine/threonine-rich polypeptide containing a P-loop motif and an SH3-binding region with phosphorylation sites for a variety of protein kinases (cdc2, CDK2, MAPK, CDK5, protein kinase C, Ca(2+)/calmodulin protein kinase 2, casein kinase 2) involved in cell proliferation and differentiation. Immunohistochemistry revealed that during early development, expression was associated with proliferating and migrating cells throughout the rodent brain, initially appearing in the proliferative ventricular zones. During late development and in adult human brain, CROC-4 was expressed in diverse brain regions including the thalamus, subthalamic nucleus, corpus callosum, substantia nigra, caudate nucleus, amygdala, and hippocampus. The association of CROC-4 expression with proliferating regions of developing brain and retention in regions of the adult brain, as well as the punctate nuclear location, suggest that CROC-4 participates in brain-specific c-fos signaling pathways involved in cellular remodeling of brain architecture.

Purification of chick retinal ganglion cells for molecular analysis: combining retrograde labeling and immunopanning yields 100% purity.

Retinal ganglion cells (RGCs) from embryonic and posthatch chickens were 100% purified by a novel combination of three steps: (1) Retrograde labeling by injection of the fluorescent carbocyanine tracer DiI into the optic nerve, (2) immunopanning of dissociated retinal cells with Thy1 antibodies, and (3) micro-aspiration of labeled RGCs into glass capillaries. The retina was dissected and dissociated with trypsin 12-15 h after the injection of DiI. DiI-labeled cells were identified on immunopanned dishes by fluorescence and collected for molecular analysis within 3 h after dissociation. This technique allowed the collection of up to 500 RGCs per capillary tube and 1500 labeled RGCs per retina. Extraction of RNA and molecular analysis by RT-PCR from 600 RGCs shows that expression of rare genes, such as those of neurotrophic factors, can be detected. This is the first description of a rapid and reliable technique for a 100% purification of RGCs with sufficient yield for molecular analysis of rare gene expression. The protocol can be modified for the purification of other cell types. The advantages and limitations of the three-step purification method are compared with previous RGC purification protocols.

Expression of doublecortin correlates with neuronal migration and pattern formation in diverse regions of the developing chick brain.

The development of functional layers in the brain involves spatially and temporally regulated gene expression. Through cDNA library screening, we have identified genes that are expressed in a neural-specific manner during brain development. Sequencing and expression data indicate that one of the clones, 18C15, is the chick homologue of doublecortin, a human X-linked gene found to be mutated in subcortical laminar heterotopia (double cortex syndrome) and lissencephaly. The 18C15 mRNA contains multiple motifs that are known to regulate mRNA stability in response to inductive signals, and these motifs are conserved between the chick and human sequences. Doublecortin is found to be expressed at peak levels during early development of the cerebellum and forebrain, and is expressed in other regions including the tectum, spinal cord, and dorsal root ganglia. This study demonstrates both spatial and temporal regulation of doublecortin expression in the chick, which is associated with early events in brain development, including neuronal migration.

Glutamate transport in cultures from developing avian cerebellum: presence of GLT-1 immunoreactivity in Purkinje neurons.

Immunocytochemical studies indicated that Purkinje cells cultured from chick embryonic cerebellum (embryonic day 8) strongly express a glutamate transporter EAAT2 cloned from human brain (GLT-1 in rat brain). At both 7 days and 14 days in culture, Purkinje neurons accumulated 1 microM [3H]L-glutamate via a potent "high-affinity" transport system that could be inhibited by D- and L-threo-3-hydroxyaspartate (D- and L-t-3OHA) and by L-trans-pyrrolidine-2,4-dicarboxylate (L-t-PDC). The order of potency of the three inhibitors was L-t-PDC approximately L-t-3OHA > D-t-30HA. Only the value of IC50 (concentration causing 50% inhibition) for D-t-3OHA significantly changed between 7 days (116 microM) and 14 days in culture (40 microM). All nH approximately 1, except in the case of the inhibition by D-t-3OHA at 14 days in culture (nH = 0.57), indicating the possible appearance of heterogeneity of the transport sites at later stages of culturing. Chronic inhibition of L-glutamate transport by L-t-PDC resulted in major changes in the morphology of Purkinje cells; particularly, the neurites almost completely regressed.

Structural compartments within neurons: developmentally regulated organization of microfilament isoform mRNA and protein.

The microfilament system is thought to be a crucial cytoskeletal component regulating development and mature function of neurons. The intracellular distribution of the microfilament isoform components, actin and tropomyosin (Tm), in neurons primarily in vivo, has been investigated at both the mRNA and the protein level using isoform specific riboprobes and antibodies. Our in vivo and in vitro studies have identified at least six neuronal compartments based on microfilament isoform mRNA localization: the developing soma, the mature soma, growth cone, developing axon hillock/proximal axon, mature somatodendritic and mature axonal pole soma. Protein localization patterns revealed that the isoforms were frequently distributed over a wider area than their respective mRNAs, suggesting that isoform specific patterns of mRNA targeting may influence, but do not absolutely determine, microfilament isoform location. Tm4 and Tm5 showed identical mRNA targeting in the developing neuron but distinct protein localization patterns. We suggest that in this instance mRNA location may best be viewed as a regulated site of synthesis and assembly, rather than a regulator of protein localization per se. In addition, Tm5 and beta-actin mRNA and protein locations were developmentally regulated, suggesting the possibility that environmental signals modulate targeting of specific mRNAs and their proteins. Thus, developmentally regulated mRNA localization and positional translation may act in concert with protein transport to regulate neuronal microfilament composition and consequently neuronal structure.

Splicing of two internal and four carboxyl-terminal alternative exons in nonmuscle tropomyosin 5 pre-mRNA is independently regulated during development.

Four nonmuscle tropomyosin isoforms have been reported to be produced from the rat Tm5 gene by alternative splicing (Beisel, K. W., and Kennedy, J. E. (1994) Gene (Amst.) 145, 251-256). In order to detect additional isoforms that might be expressed from that gene, we used reverse transcriptase-polymerase chain reaction assays and evaluated the presence of all product combinations of two alternative internal exons (6a and 6b) and four carboxyl-terminal exons (9a, 9b, 9c, and 9d) in developing and adult rat brain. We identified five different combinations for exon 9 (9a + 9b, 9a + 9c, 9a + 9d, 9c, and 9d), and the exon combinations 9a + 9c and 9a + 9d were previously unreported. Each of these combinations existed with both exon 6a and exon 6b. Thus, the rat brain generates at least 10 different isoforms from the Tm5 gene. Northern blot hybridization with alternative exon-specific probes revealed that these isoforms were also expressed in a number of different adult rat tissues, although some exons are preferentially expressed in particular tissues. Studies of regulation of the 10 different Tm5 isoform mRNAs during rat brain development indicated that no two isoforms are coordinately accumulated. Furthermore, there is a developmental switch in the use of exon 6a to exon 6b from embryonic to adult isoforms. TM5 protein isoforms show a differential localization in the adult cerebellum.

Thy-1 and AvGp50 signal transduction complex in the avian nervous system: c-Fyn and G alpha i protein association and activation of signalling pathways.

We have previously reported the isolation of two distinct populations of detergent resistant membrane complexes (DRMC's) from day-old chick brain (Henke et al.: J Neurosci Res 45:617-630, 1996). We now show that the glycosylphosphatidylinositol-anchored proteins Thy-1 and AvGp50 are associated in a signalling complex with c-Fyn, the heterotrimeric G alpha i subfamily members G alpha i-3, G alpha z, and G alpha o, alpha and beta tubulin, and a number of other phosphoproteins in immunocomplexes isolated from both populations of DRMC's. Activation of this signalling complex via Thy-1 monoclonal antibody incubation with chick forebrain cells, elicited a decrease in total phosphoprotein profile and tyrosine kinase activity present in DRMC fractions isolated from these cells, while AvGp50 and control antibodies had no effect. Down-regulation of the DRMC phosphoprotein profile was accompanied by an increase in the Thy-1-associated signalling complex, suggesting that activation of this complex initiates the decreases seen in overall DRMC kinase activity. This signalling complex provides the basis for GPI-anchored protein-mediated signal transduction within the unique plasma membrane domains represented by DRMC's.

AvGp50, a predominantly axonally expressed glycoprotein, is a member of the IgLON's subfamily of cell adhesion molecules (CAMs).

We have previously reported a 50 kDa glycoprotein (AvGp50) expressed specifically in the chick nervous system [Hancox, K.A., Sheppard, A.M. and Jeffrey, P.L., Characterisation of a novel glycoprotein (AVGP50) in the avian nervous system, with a monoclonal antibody, Dev. Brain Res., 70 (1992) 25-37], and we present its molecular characterization. A PCR fragment was generated following sequencing of peptide and N-terminal fragments derived from purified AvGp50. A 1.58 kb clone (pUEX762) containing the 5'-UTR, the entire coding sequence and a short 3'-UTR was then isolated from a chick embryonic day 18 forebrain library. The deduced amino acid sequence encodes a 338 amino acid peptide containing a 31 amino acid signal peptide at the N-terminal and a 19 amino acid phosphatidylinositol glycan linkage sequence at the C-terminal. The mature protein contains three C2-immunoglobulin-like domains and a glycosyl phosphatidylinositol anchor and shares significant homology to other members of the immunoglobulin superfamily, including neural cell adhesion molecule (N-CAM), myelin-associated glycoprotein (MAG) and the Drosophila protein Amalgam. AvGp50 exhibits highest sequence identity to a recently classified subgroup of the immunoglobulin superfamily (IgLONs - immunoglobulin LAMP, OBCAM and neurotrimin - classified by Pimenta et al. [Pimenta, A.F., Zhukareva, V., Barbe, M.F., Reinoso, B.S., Grimley, C., Henzel, W., Fischer, I. and Levitt, P., The limbic system-associated membrane protein is an Ig superfamily member that mediates selective neuronal growth and axon targeting, Neuron, 15 (1995) 287-297], comprising the opioid binding cell adhesion molecule (OBCAM), neurotrimin and the limbic system-associated membrane protein (LAMP) suggesting that AvGp50 is a member of this subgroup. AvGp50 is expressed predominantly on the cell surface of axons, in particular Purkinje cell and granule cell axons in the cerebellum. In cerebellar and forebrain neuronal cultures, protein expression is exclusively located at the cell surface. Despite its cell surface localization, AvGp50 does not directly influence the outgrowth of neurons from explant cultures from ED8 to ED10 chick forebrain, prompting the suggestion that AvGp50 may act in later maturational events.

Tropomyosin localization reveals distinct populations of microfilaments in neurites and growth cones.

The functional and structural differences between neurites and growth cones suggests the possibility that distinct microfilament populations may exist in each domain. Tropomyosins are integral components of the actin-based microfilament system. Using antibodies which detect three different sets of tropomyosin isoforms, we found that the vast majority of tropomyosin was found in a microfilament-enriched fraction of cultured cortical neurons, therefore enabling us to use the antisera to evaluate compositional differences in neuritic and growth cone microfilaments. An antibody which reacts with all known nonmuscle isoforms of the alpha Tms gene (Tm5NM1-4) stains both neurites and growth cones, whereas a second antibody against the isoform subset, Tm5NM1-2, reacts only with the neurite. A third antibody which reacts with the Tm5a/5b isoforms encoded by a separate gene from alpha Tms was strongly reactive with both neurites and growth cones in 16-h cultures but only with the neurite shaft in 40-h cultures. Treatment of neurons with cytochalasin B allowed neuritic Tm5NM1-2 to spread into growth cones. Removal of the drug resulted in the disappearance of Tm5NM1-2 from the growth cone, indicating that isoform segregation is an active process dependent on intact microfilaments. Treatment of 40-h cultures with nocodazole resulted in the removal of Tm5NM1-2 from the neurite whereas Tm5a/5b now spread back into the growth cone. We conclude that the organization of Tm5NM1-2 and Tm5a/5b in the neurite is at least partially dependent on microtubule integrity. These results indicate that tropomyosin isoforms Tm5NM1-2, Tm5NM3-4, and Tm5a/5b mark three distinct populations of actin filaments in neurites and growth cones. Further, the composition of microfilaments differs between neurites and growth cones and is subject to temporal regulation.

Characterization of two distinct populations of detergent resistant membrane complexes isolated from chick brain tissues.

We report the isolation of two distinct populations of detergent resistant membrane complexes (DRMCs) from 1-day-old chick brain, utilizing a procedure involving Triton X-100 insolubility and sucrose density gradient centrifugation. The first population is abundant (1.8% of the total homogenate protein), highly enriched for two glycosylphosphatidylinositol (GPI)-anchored proteins (Thy-1 and AvGp50), and not enriched for caveolin. The second population is of relatively low abundance (0.2% of the total homogenate), contains relatively low levels of Thy-1 and AvGp50 enrichment, and is highly enriched in caveolin. Both populations of DRMCs are enriched for cholesterol, ganglioside GM1, total kinase and tyrosine kinase activities, and c-Src and c-Fyn. However, there are differences in the Coomassie-stained protein profiles, phosphoprotein components, tyrosine kinase activity, and electron microscopic morphology when the Thy-1 and AvGp50-enriched DRMCs are compared to the caveolin-rich DRMCs. In addition, the GPI-enriched DRMCs contain CaM kinase type II immunoreactivity, whereas this molecule was undectable in the caveolin-rich DRMCs. The isolation of two distinct DRMC fractions may be representative of unique plasma membrane signaling domains involved in GPI-anchored protein or other receptor-mediated signal transduction within the avian nervous system. Further, we have demonstrated for the first time that nervous system tissue, in particular the hatch chick cerebellum, contains caveolin immunoreactivity.

Epigenetic factors controlling the development of avian Purkinje neurons.

Using a unique protocol, we have developed an avian neuron culture system in which a high yield of Purkinje neurons is obtained more readily than with pre-existing methods. Purkinje neurons were identified in vitro using the specific antibodies calbindin and cyclic GMP-kinase. Survival of Purkinje neurons was dependent on astrocyte contact and enhanced by astrocytic factors supplied to the medium by a monolayer of astrocytes grown on coated membranes suspended in the culture wells but not in contact with the neurons. The age of the cerebellum from which astrocytes were obtained was shown to affect the morphological development of the Purkinje neurons suggesting the developmentally-regulated expression of growth factors. However, in the presence of the astrocytes, Purkinje neurons could only progress to a limited stage of development based on morphological criteria. The addition to the culture of cerebellar granule neurons at a time of Purkinje neuron development that they would expect to encounter them in vivo resulted in a shift of Purkinje neurons to a mature phenotype. This maturation effect was increased in response to increasing levels of granule neurons, but was independent of the granule neuron ages used. This system offers significant advantages over other Purkinje neuron culture systems and will be useful for studying the extrinsic factors involved in Purkinje neuron development and histogenesis.

Differential induction and intracellular localization of SCG10 messenger RNA is associated with neuronal differentiation.

The differentiation of neurons involves the establishment of distinct molecular compartments which regulate neuronal shape and function. This requires targeting of specific gene products to growth-associated regions of the neuron. We have investigated the temporal and spatial regulation of SCG10 gene expression during neuronal differentiation. There are two SCG10 messenger RNAs, 1 and 2 kg in length, which encode the same growth-associated protein. These messenger RNAs were found to be differentially regulated during the onset of neurite outgrowth in early rat cerebellum development. In PC12 cells, the two SCG10 messenger RNAs were shown to be differentially induced by nerve growth factor. Regulation of the 2 kb messenger RNA, but not the 1 kb messenger RNA, is dependent on the differentiation of PC12 cells, indicating that post-transcriptional regulation of SCG10 expression during neurite outgrowth. Spatial regulation of the 2 kb SCG10 messenger RNA distribution during brain development was examined by in situ hybridization. The 2 kb messenger RNA was found to be localized to the neuronal pole where outgrowth was occurring, within differentiating neurons in vivo. Intracellular localization of SCG10 messenger RNA was also observed in differentiating primary cultured neurons, with the 2 kb messenger RNA transported into growing neurites during the development of neuronal polarity. In neurons which had developed polarity, the 2 kb SCG10 messenger RNA was consistently found in the cell body and axon. This study demonstrates both temporal and spatial post-transcriptional regulation of SCG10 expression which is associated with neurite outgrowth. The directed transport and positional translation of SCG10 messenger RNA provide a potential mechanism for protein targeting and the creation of molecular compartments during neuronal differentiation.

Intracellular localization of tropomyosin mRNA and protein is associated with development of neuronal polarity.

Neuronal differentiation involves extensive rearrangement of the cytoskeleton, including the actin-based microfilament system, and establishment of molecular compartments within the neuron. The intracellular distribution of tropomyosin (Tm) mRNA in vivo and in vitro has been examined and correlated with protein targetting. The mRNAs encoding two Tm isoforms were found to be differentially localized in developing neurons. Tm-5 mRNA is localized to the axonal pole of differentiating embryonic rat neurons, in contrast to TmBr-2 mRNA distribution throughout the cell body. Tm-5 mRNA is transported into the axon of differentiating primary cultured neurons. This mRNA localization is developmentally regulated and correlates with the targeting of Tm-5 protein to growing axons. Tm-5 colocalizes with a subset of neuronal microfilaments associated with the initiation and maintenance of outgrowth. The segregation of Tm-5 is the earliest known marker of neuronal polarity and may play a role in the establishment of polarity.

Induction of neuron-specific tropomyosin mRNAs by nerve growth factor is dependent on morphological differentiation.

We have examined the expression of brain-specific tropomyosins during neuronal differentiation. Both TmBr-1 and TmBr-3 were shown to be neuron specific. TmBr-1 and TmBr-3 mRNA levels increased during the most active phase of neurite outgrowth in the developing rat cerebellum. In PC12 cells stimulated by nerve growth factor (NGF) to differentiate to the neuronal phenotype, TmBr-1 and TmBr-3 levels increased with an increasing degree of morphological differentiation. Induction of TmBr-1 and TmBr-3 expression only occurred under conditions where PC12 cells were permitted to extend neurites. NGF was unable to maintain levels of TmBr-1 and TmBr-3 with the loss of neuronal phenotype by resuspension of differentiated PC12 cells. The unique cellular expression and regulation in vivo and in vitro of TmBr-1 and TmBr-3 strongly suggests a critical role of these tropomyosins in neuronal microfilament function. The findings reveal that the induction and maintenance of the neuronal tropomyosins is dependent on morphological differentiation and the maintenance of the neuronal phenotype.

Characterisation of a novel glycoprotein (AvGp50) in the avian nervous system, with a monoclonal antibody.

A size fractionated lentil lectin-positive fraction derived from a deoxycholate extract of 1-day-old chick forebrain membranes was used to generate a series of monoclonal antibodies (Mabs) against neural antigens. One of these, MabSA1.7 recognises a glycoprotein which is enriched in synaptic plasma membranes, designated AvGp50. Polyacrylamide gel electrophoresis and Western blots show that AvGp50 is comprised of at least two glycoforms, with M(r)s of 53 kDa and 49 kDa respectively. AvGp50 is nervous system specific and most abundantly expressed in the forebrain, tecta and cerebellum where its pattern of expression is developmentally regulated. Immunohistochemical data localises AvGp50 to regions characterised by highly concentrated synapses, in particular, the molecular and granule cell layers of the cerebellum and in the inner and outer plexiform layers in the retina. Solubilization of the protein with the detergent Triton X-100 shows that AvGp50 is predominantly a cytoskeletally associated glycoprotein. However, when a synaptic plasma membrane fraction was treated with Triton X-114, AvGp50 partitioned into the detergent phase. Digestion of the protein with N-glycanase cleaved five N-linked carbohydrate side chains and reduced the molecular weight to approximately 34 and 31 kDa. Removal of the carbohydrate side chains led to an almost complete loss of recognition of the 34 kDa glycoform by the MabSA1.7, suggesting that the monoclonal antibody recognises a carbohydrate rather than peptide epitope.

Molecular cloning and primary structure of the avian Thy-1 glycoprotein.

We have previously characterised, both biochemically and immunohistochemically, a 23 kDa putative avian Thy-1 protein homologue. In this report we have examined the carbohydrates present on the protein and determined the partial protein sequence of enzymatically and CNBr-produced peptides. The protein sequences enabled us to clone an essentially full-length (1854 bp) cDNA using PCR and colony screening of an embryonic day (ED) 18 forebrain pUEX-1 cDNA library. Analysis of deduced amino acid sequence shows the 23 kDa protein to be 110 amino acids in length compared to mouse (112) and human and rat (111) while still retaining the conserved cysteine residues. The N-glycosylation site at position 61 is the same as that in the human protein, but is different from that in the rodent (position 75). Northern blot analysis of Thy-1 mRNA expression in the forebrain, cerebellum and tectum show that all three tissues have low levels at ED4 (forebrain and tectum) and ED8 (cerebellum), rising most rapidly between ED16 and the first few days post-hatch. Analysis of various tissues at hatch and adult show expression to be predominantly neuronal with very low levels in some other organs, mainly at hatch, indicating the possibility of subsets of cells, which we have also seen in histological sections, in these tissues expressing Thy-1 mRNA. Bone marrow and blood cells were also negative for Thy-1 protein.

Denervation-induced changes in lectin binding to sarcolemmal glycoproteins: exposure of cryptic recognition sites.

The increase in Concanavalin A (ConA) binding to sarcolemmal membranes of rat skeletal muscle following denervation has been attributed to conformational changes in membrane glycoproteins resulting in the unmasking of previously cryptic ConA binding sites (Leung et al., 1982). In this study, analysis of lectin binding patterns to alpha-fucosidase- or sialidase-treated sarcolemmal membranes reveals that the fucose moieties of carbohydrate structures may be principally involved in the unmasking process. By contrast, sialic acid has no apparent effect on the availability of the number of ConA binding sites, but plays a significant role in the masking of other lectin recognition sites.

Expression of actin and myosin genes during PC12 cell differentiation.

We have measured the accumulation of transcripts for myosin and actin during NGF induced differentiation of PC12 cells. Beta (beta) and gamma (gamma) actin and myosin light chains (MLC) 2 and 3 show different patterns of expression, with transient elevations in gene expression one day after NGF addition. This elevation occurs earlier than that of neurite outgrowth, neurofilament protein (NF68) (16) and Thy-1 glycoprotein gene expression. These results suggest differing mechanisms of control of actin and myosin expression, together with a varying function and relationship between them during NGF-induced neurite differentiation.

Thy-1 antigen is specific to ganglion cells in chicks.

The cellular localization of Thy-1 in the chick retina was investigated by selectively destroying certain populations of neurons with toxins. In control retinae four weeks after intravitreal injection of vehicle, there was strong immunoreactivity for Thy-1 in the nerve fibre layer, ganglion cell layer and inner plexiform layer. By contrast, 4 weeks after intraocular injection with 1.25 nmol of colchicine, virtually all ganglion cells had been destroyed, but most amacrine cells remained. Very little Thy-1 immunoreactivity was evident in these retinae. Four weeks after intraocular injection of 2 mumol of N-methyl-D-aspartic acid (NMDA), a large proportion of amacrine cells had been destroyed, but most ganglion cells remained. In these retinae Thy-1 immunoreactivity was present in the nerve fibre, ganglion cell and inner plexiform layers, in the latter with greater intensity than in controls. We conclude that in chicks the Thy-1 antigen is principally, if not exclusively restricted to ganglion cells.