Shin Takagi

Spatial and temporal expression pattern of N-cadherin cell adhesion molecules correlated with morphogenetic processes of chicken embryos☆

N-cadherin is a Ca2+-dependent cell-cell adhesion molecule, which was identified in brain cells of mouse and chicken. In the present study, we have determined the pattern of expression of N-cadherin in chicken embryos at various stages by means of immunohistochemistry. N-cadherin was expressed in cells derived from all three primary germ layers. Its expression was transient in many tissues but permanent in others. The transient expression occurred in nephric tubules, skeletal muscles, mesenchymal tissues, endodermal organs, and epidermis, while the permanent expression occurred in nervous systems, lens, and myocardiac cells. Appearance or disappearance of N-cadherin could be generally correlated with morphogenetic events, such as rearrangement, segregation, or association of cells. Comparison of the expression pattern of N-cadherin with that of L-CAM and N-CAM determined by other workers suggests that there is some mechanism controlling expression of multiple classes of adhesion molecules. The pattern of expression of N-cadherin was generally complementary to that of L-CAM; that is, if N-cadherin appeared, L-CAM disappeared or vice versa. We also found cases in which N-cadherin was expressed in the same local regions as L-CAM. The distribution of N-cadherin was similar to that of N-CAM with some exceptions. Thus, N-cadherin and other cell-cell adhesion molecules seem to be expressed under a precise spatial and temporal control so as to be associated with a variety of morphogenetic events during development.

Developmentally regulated expression of a cell surface protein, neuropilin, in the mouse nervous system

Neuropilin (previously A5) is a cell surface glycoprotein that was originally identified in Xenopus tadpole nervous tissues. In Xenopus, neuropilin is expressed on both the presynaptic and postsynaptic elements in the visual and general somatic sensory systems, suggesting a role in neuronal cell recognition. In this study, we identified a mouse homologue of neuropilin and examined its expression in developing mouse nervous tissues. cDNA cloning and sequencing revealed that the primary structure of the mouse neuropilin was highly similar to that of Xenopus and that the extracellular segment of the molecule possessed several motifs that were expected to be involved in cell-cell interaction. Immunohistochemistry and in situ hybridization analyses in mice indicated that the expression of neuropilin was restricted to particular neuron circuits. Neuropilin protein was localized on axons but not on the somata of neurons. The expression of neuropilin persisted through the time when axons were actively growing to form neuronal connections. These observations suggest that neuropilin is involved in growth, fasciculation, and targeting for a particular groups of axons.

The A5 antigen, a candidate for the neuronal recognition molecule, has homologies to complement components and coagulation factors

The A5 antigen is a neuronal cell surface protein of Xenopus presumed to be involved in the neuronal recognition between the optic nerve fibers and the visual centers. Analyses of cDNA clones revealed that the A5 antigen is a class I membrane protein containing two different internal repeats in the extracellular segment. The first repeat bears homology to domain III of complement components C1r and C1s, and the second repeat is homologous to the C1 and C2 domains of coagulation factors V and VIII. The mRNA for the A5 antigen was present in retinal ganglion cells and visual center neurons. Nonneuronal cells in the peripheral and central nervous systems did not express the mRNA for the A5 antigen.

Specific cell surface labels in the visual centers of Xenopus laevis tadpole identified using monoclonal antibodies

Monoclonal antibodies (MAbs) against the optic tectum of Xenopus tadpoles were generated and screened by the immunofluorescent staining of frozen sections of tadpole brains. MAb-A5 stains the 8th and 9th plexiform layers of the optic tectum, whereas MAb-B2 stains all but the eighth and ninth plexiform layers of the optic tectum. MAb-A5 antigen is also detectable in the nucleus of Belonci, the corpus geniculatum thalamicum, the pretectal area, and the basal optic nucleus, all targets of the optic nerve, but is not detectable in the optic nerve or the optic tract. On the other hand, MAb-B2 does not stain any of these visual centers, though many fibers surrounding them are stained. Eye-enucleation experiments showed that MAb-A5 antigen is expressed in the optic tectum even when it is not innervated by optic nerves. Staining of viable brains with these MAbs indicates that these antigens are cell surface molecules. Immunoadsorption followed by SDS-PAGE suggests that proteins are constituents of these antigens. The MAb-A5 antigen in the diencephalon and the mesencephalon is not detectable at stage 35/36, but is detectable at stage 39 when the optic nerves begin to innervate the optic tectum. The spatial as well as the temporal patterns of the expression of the MAb-A5 antigen suggest that this molecule may be involved in the target recognition of optic nerve fibers.

A lymphocyte‐specific CC chemokine, secondary lymphoid tissue chemokine (SLC), is a highly efficient chemoattractant for B cells and activated T cells

Secondary lymphoid tissue chemokine (SLC) is a CC chemokine expressed mainly in lymphnodes, appendix and spleen, and specifically chemotactic for lymphocytes (Nagira et al., J. Biol. Chem. 1997. 272: 19518 – 19524). Here, we carried out transendothelial migration assays to determine the classes and subsets of lymphocytes migrating toward SLC. SLC attracted freshly isolated B cells with high efficiency and T cells modestly. Thus, SLC is the first CC chemokine with a strong chemotactic activity on fresh B cells. Among T cell types and subsets, SLC broadly attracted CD4+ and CD8+ cells, CD45RO– (naive) and CD45RO+ (memory) cells, and CD26high (activated) and CD26low/− (resting) cells. SLC also attracted both L‐selectin+ and L‐selectin– subpopulations of various T cell subsets and B cells. Furthermore, mitogenic stimulation strongly enhanced migratory responses of T cells and B cells toward SLC. By in situ hybridization, SLC mRNA was detected in the cortical parafollicular regions (the T cell areas) of a lymph node and an appendix. Collectively, SLC may be a basic chemokine supporting homeostatic migration of a broad spectrum of lymphocytes into the secondary lymphoid tissues. SLC may also be involved in immune responses by inducing highly efficient migration of T and B cells following antigenic stimulation.

Identification of Single C Motif-1/Lymphotactin Receptor XCR1

Single C motif-1 (SCM-1)/lymphotactin is a member of the chemokine superfamily, but retains only the 2nd and 4th of the four cysteine residues conserved in other chemokines. In humans, there are two highly homologous SCM-1 genes encoding SCM-1α and SCM-1β with two amino acid substitutions. To identify a specific receptor for SCM-1 proteins, we produced recombinant SCM-1α and SCM-1β by the baculovirus expression system and tested them on murine L1.2 cells stably expressing eight known chemokine receptors and three orphan receptors. Both proteins specifically induced migration in cells expressing an orphan receptor, GPR5. The migration was chemotactic and suppressed by pertussis toxin, indicating coupling to a Gα type of G protein. Both proteins also induced intracellular calcium mobilization in GPR5-expressing L1.2 cells with efficient mutual cross desensitization. SCM-1α bound specifically to GPR5-expressing L1.2 cells with a K d of 10 nm. By Northern blot analysis, GPR5 mRNA of about 5 kilobases was detected strongly in placenta and weakly in spleen and thymus among various human tissues. Identification of a specific receptor for SCM-1 would facilitate our investigation on its biological function. Following the set rule for the chemokine receptor nomenclature, we propose to designate GPR5 as XCR1 from XC chemokine receptor-1.

Plexin: A novel neuronal cell surface molecule that mediates cell adhesion via a homophilic binding mechanism in the presence of calcium ions

Plexin (previously referred to as B2) is a neuronal cell surface molecule that has been identified in Xenopus. cDNA cloning reveals that plexin has no homology to known neuronal cell surface molecules but possesses, in its extracellular segment, three internal repeats of cysteine clusters that are homologous to the cysteine-rich domain of the c-met proto-oncogene protein product. The exogenous plexin proteins expressed on the surfaces of L cells by cDNA transfection mediate cell adhesion via a homophilic binding mechanism, under the presence of calcium ions. Plexin is expressed in the receptors and neurons of particular sensory systems. These findings indicate that plexin is a novel calcium-dependent cell adhesion molecule and suggest its involvement in specific neuronal cell interaction and/or contact.

Infrared laser-mediated gene induction in targeted single cells in vivo.

We developed infrared laser–evoked gene operator (IR-LEGO), a microscope system optimized for heating cells without photochemical damage. Infrared irradiation causes reproducible temperature shifts of the in vitro microenvironment in a power-dependent manner. When applied to living Caenorhabditis elegans, IR-LEGO induced heat shock–mediated expression of transgenes in targeted single cells in a more efficient and less deleterious manner than a 440-nm dye laser and elicited physiologically relevant phenotypic responses.

ROLES OF A NEURONAL CELL-SURFACE MOLECULE, NEUROPILIN, IN NERVE FIBER FASCICULATION AND GUIDANCE

Abstract. Neuropilin is a cell-surface glycoprotein that was first identified in Xenopus tadpole nervous tissues and then in chicken and mouse. The primary structure of neuropilin is highly conserved among these vertebrate species. The extracellular part of the molecule is composed of three domains referred to as a1/a2, b1/b2, and c, each of which is expected to be involved in molecular and/or cellular interactions. Neuropilin can mediate cell adhesion by heterophilic molecular interaction. In all vertebrate species examined, the neuropilin protein is restricted to axons of particular neuron classes, and at stages when axon growth is active. The gain and loss of function of neuropilin in developing mouse embryos causes defasciculation and incorrect sprouting of nerve fibers. These findings suggest that neuropilin serves in a variety of neuronal cell interactions by binding to a variety of molecules, and that it plays essential roles in nerve fiber fasciculation and guidance.

A diurnal variation of vasoactive intestinal peptide (VIP) mRNA under a daily light-dark cycle in the rat suprachiasmatic nucleus

SummaryWe detected a diurnal change of vasoactive intestinal peptide (VIP) messenger RNA (mRNA) in the rat suprachiasmatic nucleus (SCN) using the in situ hybridization technique combined with computed image analysis. The amount of VIP mRNA was greatest at 02.00 h, the next largest level was seen at 20.00 h during the dark phase and smallest at 14.00 h during the light phase. We also confirmed that the onset of the fluctuation of mRNA occurred 2–6 h prior to the diurnal variation of the content of VIP-like immunoreactivity by semiquantitative immunocytochemistry. These findings suggest that light stimulus from the retina is a primarily influence on the transcription of VIP mRNA and induces a diurnal variation of VIP synthesis.