Abstract. Several members of the Ig superfamily are expressed on neural cells where they participate in surface interactions between cell bodies and processes. Their Ig domains are more closely related to each other than to Ig variable and constant domains and have been grouped into the C2 set. Here, we report the cloning and characterization of another member of this group, the mouse neuronal cell surface antigen F3. The F3 eDNA sequence contains an open reading frame that could encode a 1,020-amino acid protein consisting of a signal sequence, six Ig-like domains of the C2 type, a long premembrane region containing two segments that exhibit sequence similarity to fibronectin type III repeats and a moderately hydrophobic COOH-terminal sequence. The protein does not contain a typical transmembrane segment but appears to be attached to the membrane by a phosphatidylinositol anchor. Antibodies against the F3 protein recognize a prominent 135-kD protein in mouse brain. In fetal brain cultures, they stain the neuronal cell surface and, in cultures maintained in chemically defined medium, most prominently neurites and neurite bundles. The mouse f3 gene maps to band F of chromosome 15. The gene transcripts detected in the brain by F3 cDNA probes are developmentally regulated, the highest amounts being expressed between 1 and 2 wk after birth.The F3 nucleotide and deduced amino acid sequence show striking similarity to the recently published sequence of the chicken neuronal cell surface protein contactin. However, there are important differences between the two molecules. In contrast to F3, contactin has a transmembrane and a cytoplasmic domain. Whereas contactin is insoluble in nonionic detergent and is tightly associated with the cytoskeleton, about equal amounts of F3 distribute between buffer-soluble, nonionic detergent-soluble, and detergent-insoluble fractions. Among other neural cell surface proteins, F3 most resembles the neuronal cell adhesion protein L1, with 25 % amino acid identity between their extracellular domains. Based on its structural similarity with known cell adhesion proteins of nervous tissue and with L1 in particular, we propose that F3 mediates cell surface interactions during nervous system development.

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Early B-cell factor 2 (EBF2) is one of four mammalian members of an atypical helix-loop-helix transcription factor family (COE). COE proteins have been implicated in various aspects of nervous and immune system development. We and others have generated and described mice carrying a null mutation of Ebf2, a gene previously characterized in the context of Xenopus laevis primary neurogenesis and neuronal differentiation. In addition to deficits in neuroendocrine and olfactory development, and peripheral nerve maturation, Ebf2 null mice feature an ataxic gait and obvious motor deficits associated with clear-cut abnormalities of cerebellar development. The number of Purkinje cells (PCs) in the Ebf2 null is markedly decreased, resulting in a small cerebellum with notable foliation defects, particularly in the anterior vermis. We show that this stems from the defective migration of a molecularly defined PC subset that subsequently dies by apoptosis. Part of the striped cerebellar topography is disrupted due to cell death and, in addition, many of the surviving PCs, that would normally adopt a zebrin II-negative phenotype, transdifferentiate to Zebrin II-positive, an unprecedented finding suggesting that Ebf2 is required for the establishment of a proper cerebellar cortical map.

F3/contactin (CNTN1) and TAG-1 (CNTN2) are closely related axonal glycoproteins that are differentially regulated during development. In the cerebellar cortex TAG-1 is expressed first as granule cell progenitors differentiate in the premigratory zone of the external germinal layer. However, as these cells begin radial migration, TAG-1 is replaced by F3/contactin. To address the significance of this differential regulation, we have generated transgenic mice in which F3/contactin expression is driven byTAG-1 gene regulatory sequences, which results in premature expression of F3/contactin in granule cells. These animals (TAG/F3mice) display a developmentally regulated cerebellar phenotype in which the size of the cerebellum is markedly reduced during the first two postnatal weeks but subsequently recovers. This is due in part to a reduction in the number of granule cells, most evident in the external germinal layer at postnatal day 3 and in the inner granular layer between postnatal days 8 and 11. The reduction in granule cell number is accompanied by a decrease in precursor granule cell proliferation at postnatal day 3, followed by an increase in the number of cycling cells at postnatal day 8. In the same developmental window the size of the molecular layer is markedly reduced and Purkinje cell dendrites fail to elaborate normally. These data are consistent with a model in which deployment of F3/contactin on granule cells affects proliferation and differentiation of these neurons as well as the differentiation of their synaptic partners, the Purkinje cells. Together,these findings indicate that precise spatio-temporal regulation of TAG-1 and F3/contactin expression is critical for normal cerebellar morphogenesis.

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The transmembrane orientation of the polypeptide chains present in preparations of adult and neonatal mouse N-CAM was studied using, as a model system, liposome-inserted purified N-CAM preparations. N-CAM purified from adult or neonatal mouse brain was 1251-labeled and reconstituted into artificial lipid vesicles. After trypsin digestion, the peptides thar remained associated with the liposomes were isolated by floatation of the vesicles on sucrose gradients. In control experiments the liposomes were lysed before trypsin treatment. Large, overlapping peptides were obtained after this treatment, several of which were protected by the liposome membrane. Sialic-acidbearing peptides were revealed by their sensitivity to neuraminidase. To distinguish between peptides corresponding to intracellular or extracellular domains use was made of the P61 and H28.123 monoclonal antibodies, which recognize determinants located on the cytoplasmic and the extracellular part of the molecules respectively.There was no indication that the N-CAM chains were inserted in an inside-out configuration. Peptides protected from trypsin attack by the liposomes and recognized only by P61 had M , values of 92000,42000 and 35000. The H28.123 determinant could be mapped to a 32000-M, peptide located close to the membrane at the vesicle's exterior. The bulk of the sialic acid seemed to be carried by a rather short sequence distal to the H28.123-reactive peptide but at some distance from the N terminus. Fragments of very similar M , were generated from young and adult material.However, a 45000-M, peptide from neonatal N-CAM appeared to migrate in the high-Mr region of sodium dodecyl sulfatejpolyacrylamide &ells in its fully sialylated form.It is concluded that (a) identical polypeptide chains are present in young and adult preparation, (b) the 180000-Mr, 140000-Mr and 120000-Mr chains differ by the length of their cytoplasmic extensions and (c) the largest cytoplasmic sequences have a Mr close to 90000. A tentative linear model of the transmembrane topography of the N-CAM polypeptides is presented.The neural cell adhesion molecules, called N-CAMS, are among the best-characterized cell adhesion proteins [I, 21. Unsolved questions concerning their structure are their mode of interaction with the cell membrane and the exact relationship between the two or three structurally similar polypeptides isolated from chick [3] or rodent [4 -61 brain. Our approach to these problems has been to reconstitute purified mouse N-CAM into artificial lipid vesicles and to analyze the peptides generated by trypsin digestion of these vesicles. A number of previous studies have addressed the question of how intrinsic membrane proteins insert in liposomes (see for instance [7 -9]), and it is generally agreed that the proteins interact with the vesicle bilayer in a way closely resembling their arrangement in the native membrane. However, few studies on the transmembrane topography of proteins have Abbreviations. SDS, sodium dodecyl sulfate; Tris/saline, 50 mM Tris pH 7.4,...

The cell-adhesion molecules N-CAM (neural cell-adhesion molecule) are ligands in the formation of cell-cell bonds and have been shown to play important roles during neuro-ontogenesis. They exist in several molecular forms which differ at the protein and carbohydrate levels. The regulation of the expression of these different forms is an important issue that bears on such questions as to how adhesive interactions between cells are modulated during morphogenesis. In the present study we have used N-CAM cDNA clones to investigate the expression of the cognate mRNAs in the mouse and rat brain and in 2 neural cell lines. The results were compared with the levels of the different N-CAM proteins. We made the following observations. A complex set of 5 size classes of mRNAs--which show developmental, regional, and cell-type-dependent variations in their expression--hybridize to 1 of our cDNA probes. While embryonic brain contains N-CAM gene transcripts 7.4, 6.7, and 4.3 kilobases (kb) in length, 2 additional mRNAs of 5.2 and 2.9 kb appear postnatally. Transformed brain cells of an astrocytic character express predominantly mRNAs of 6.7, 4.3, and 2.9 kb and a neuroblastoma line those of 7.4, 6.7, 4.3, and 2.9 kb. There are important quantitative changes in the amount of N-CAM message expressed during brain development, with a peak around birth, suggesting that N-CAM synthesis is controlled at the transcriptional level. A comparison of N-CAM protein and mRNA levels reveals a striking correlation between the relative concentrations of the Mr 120,000 N-CAM protein (N-CAM120) and the 5.2 kb transcript.(ABSTRACT TRUNCATED AT 250 WORDS)

The distribution of the F3/F11 neuronal cell surface molecule was investigated in the developing and adult mouse cerebellum by immunocytochemistry at the light and electron microscopic levels. F3/F11 was confined to subsets of neuronal types, since the Purkinje cell body and dendritic arborization as well as the stellate cells were not immunoreactive. In the young developing cerebellum, the granule cell axons strongly express F3/F11 as soon as they begin to grow, consistent with a functional role in promoting directional outgrowth of neuronal processes. In 10-d-old and adult cerebella, the granule cell bodies and dendrites were not immunoreactive whereas the parallel fibers, which are the granule cell axons, were labeled including in their presynaptic varicosities. By contrast, dendrites, cell bodies, and axons of Golgi cells were labeled by anti-F3 antibodies. Hence, F3/F11 can either be expressed throughout the cell or be polarized to the axons. This raises the question of how segregation of the glypiated F3/F11 molecule between different subcellular compartments depending on the type of neuron is achieved. F3/F11 was found to be present at three types of synaptic sites, suggesting that it might play a role in the formation and maintenance of synapses. However, in each type of synpase, F3/F11 was present at only the pre- or postsynaptic site, never at both: the parallel fiber varicosities contained F3/F11 whereas the postsynaptic compartment in contact, that is, the Purkinje cell dendritic spines, did not. The granule cell dendrites were unlabeled while the mossy fiber terminals contacting them were immunoreactive, and finally, the Golgi cell dendrites and dendritic spines were labeled while the presynaptic compartment contacting them was not. If F3/F11 functions as an adhesion molecule in vivo as indicated by in vitro assays, F3/F11-mediated adhesion is likely to be heterophilic.