摘要:

AbstractIn previous work, we found that the phorbol ester drug 12‐0‐tetradecanoyl phorbol acetate (TPA) reverses the developmental restriction of melanogenesis that occurs early in neural crest development, causing Schwann cell precursors to undergo a metaplastic transformation into melanocytes. In this study, we examine whether these effects of TPA may be mediated by changes in endogenous levels of protein kinase C (PKC) activities. We report that low levels of PKC activity are correlated with this adventitious pigmentation in the crest‐derived cells of dorsal root ganglia both during normal development and following TPA treatment in culture. These results suggest that regulation of endogenous levels of PKC plays a role in developmental decisions that neural crest cells make during early embryoge

ISSN:0360-4012    

DOI:10.1002/jnr.490210203

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY

摘要:

AbstractWe previously reported the production of monoclonal antibodies (Mabs) that identified cell surface components of cultured chick and quail ciliary ganglion (CG) neurons and of a subpopulation of neural crest (NC) cells from 31‐hr chick embryos (stage 9). Here we demonstrate that another Mab, CG‐14, which was prepared to nitrocellulose‐immobilized, lightly fixed (0.125% paraformaldehyde)mesencephalic NC cellsfrom 31‐hr (stage 9) chick embryos, labels the same antigen(s) recognized by CG‐1 and CG‐4 on both the CG neurons and the subpopulation of NC cells. All three Mabs label a polypeptide of 75 kD on Western blots of one‐dimensional SDS‐polyacrylamide gels. CG‐14 blocked the binding of CG‐1 and/or CG‐4 to the 75 kD band on Western blots and blocked the binding of CG‐1 and CG‐4 to CG and NC cells. CG‐1 and/or CG‐4 antibodies, in turn, blocked the binding of CG‐14 to Western blots, as well as NC and CG cells. We had previously shown that antibodies CG‐1 and CG‐4 were synergistically cytotoxic for the majority (95%) of cultured CG neurons in vitro in the presence of guinea pig complement. Here we show that the antibodies, which are both of theγ2asubclass, are also cytotoxic for the NC cells that they label in vitro. After the cells are ablated in culture, no other cells bearing the antigen(s) recognized by any of the three Mabs appear over a 2.5‐week period. CG‐14, however, is not cytotoxic for either the CG or NC cell populations alone or in combinations with CG‐1 or CG‐4.These results confirm our original observation that cultured CG neurons and NC cells share cell‐surface antigen(s). The antigen recognized by all three Mabs appears to be the same whether the immunogen used to produce the antibodies was CG neurons or NC cells. This finding encourages us to continue tests of the hypothesis that the subpopulation of mesencephalic neural crest cells contributes to the formation of the ciliary ganglion in the embryo. Further characterization of

ISSN:0360-4012    

DOI:10.1002/jnr.490210204

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY

摘要:

AbstractHigh‐affinity choline uptake mechanisms are among the characteristics of cholinergic neurons such as the ciliary and choroid subpopulations in the ciliary ganglion (Barald and Berg, 1979).We have produced three monoclonal antibodies (Mabs), two of which were made to 8‐day embryonic chick ciliary ganglion (CG) neurons (CG‐1, CG‐4) (Barald, 1982) and one of which was made to cultured mesencephalic neural crest (NC) cells (CG‐14) removed from the embryo 31 hr after incubation. We have shown that all three Mabs label a common 75 kD antigen present on the cell surface of both CG neurons and NC cells (Barald, 1988).Here we report that the CG‐1 and CG‐4 antibodies, used in the same ratios in which they are synergistically cytotoxic for both the CG and NC cells (Barald, 1988), and Mab CG‐14 alone, have specific effects on the high‐affinity choline uptake mechanism (HACU) of CG neurons and isolated antigen‐positive NC cells in the absence of complement. CG‐1 and CG‐4 in ratios of 8/1 (the same ratios that are used to kill the CG and the NC subpopulation), but neither singly, inhibit the HACU of CG neurons by 40% and that of isolated antigen‐positive NC cells by 75%. However, CG‐14 alone, at 1 μg/ml, inhibits the HACU of both CG neurons and isolated NC cells by 95%.None of the antibodies had an effect on numbers of ouabain binding sites (a measure of the Na+/K+ATPase) or cell surface acetylcholinesterase (AChE) of CG neurons or NC cells isolated by “no‐flow” fluorescence cytometry with a Meridian Instruments ACAS470 cytometer. CG or NC cells grown in the presence of the antibodies without complement grow and remain healthy for many weeks. They exhibit no difference in morphology, protein content, lactate dehydrogenase activity (LDH), or division time from untreated sister cultures.Therefore, the antigen recognized by all three Mabs may be involved in a high‐affinity choline uptake mechanism, a common characteristic of cholinergic neurons. The Mabs themselves may possibly label some element of the high‐affinity transporter or a proximal membrane component. This implies that such a high‐affinity uptake mechanism is present in the subpopulation of NC cells at early times in development. If these cells in fact are destined to contribute to the avian CG, these characteristics are present in the subpopulation before th

ISSN:0360-4012    

DOI:10.1002/jnr.490210205

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY

摘要:

AbstractTenascin is a glycoprotein associated with the extra‐cellular matrix and the surface of some cell types. Here, the distribution and possible function of tenascin have been examined along the pathways followed by cranial neural crest cells. During early stages of neural crest migration, tenascin was observed in a dense matrix surrounding premigratory cranial neural crest cells. Along the neural tube, tenascin immunoreactivity was observed in a dorsoventral gradient and was also noted under the ectoderm and around the notochord. During advanced neural crest migration, tenascin immunoreactivity colocalized with and appeared to be on the surface of migrating neural crest cells. At later stages, tenascin was present around the otic vesicles, retina, lens, and in an interstitial matrix in the region of the branchial arches. At the level of the occipital somites, tenascin immunoreactivity was observed around the neural tube, notochord, dermamyotome, and on the basal surface of the ectoderm. Tenascin was also observed in an interstitial matrix within the sclerotome. At early stages of vagal neural crest migration, immunoreactivity was uniform within the sclerotome, whereas at later stages tenascin colocalized with vagal neural crest cells within the rostral half of each sclerotome. The possible function of tenascin was tested by injecting antitenascin antibodies lateral to the mesencephalic neural tube. Two predominant defects were noted in injected embryos: (1) ectopic aggregates of cranial neural crest cells external to the neural tube and sometimes located on the apical side of the ectoderm; and (2) open and deformed neural tubes. Both the distribution and results of the perturbation experiment suggest that tenascin is required for proper cranial neural crest migratio

ISSN:0360-4012    

DOI:10.1002/jnr.490210206

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY

摘要:

AbstractAn epitope recognized by the monoclonal antibody I‐5G9 was expressed by all neural crest cells shortly after explantation into culture. At this time all neural crest cells actively migrated away from the neural tube. Immunoreactivity was localized intracellularly and organized into stress fiber‐like filaments. Often, immunofluorescence was particularly high in short fibers in the lamellipodia of the reading edge of migrating cells. Two‐week‐old cultures had a diameter of 8–10 mm. At that stage a ring of immunoreactive cells was present at the periphery of each culture, an area where cells were still migratory. An inner concentric circle had reduced and more granular staining. In this area cells had ceased to migrate. In the center of the culture cells were multilayered, nonmigratory, and did not bind I‐5G9. After creating a lesion in the nonreactive central region, some cells resumed migration into the lesioned area and reexpressed the epitope. I‐5G9 staining and phalloidin fluorescence colocalized partially in some cells and completely in others. It is concluded that the epitope recognized by I‐5G9 is expressed in a migration‐dependent manner. The partial colocalization of I‐5G9 and phalloidin fluorescence supports the notion that the epitope recognized by I‐5G9 is specifically expressed in stress fibers of migratory cells, possibly in one of the actin‐associated proteins or an F actin‐a

ISSN:0360-4012    

DOI:10.1002/jnr.490210207

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY

摘要:

AbstractWe have examined glial cell lineages during rat spinal cord development by using a variety of antibodies that react with immature and mature glia. Radial glia in embryonic cord bound (1) A2B5, an antibody that reacts with a glial precursor cell population in optic nerve; (2) AbR24, which is directed against GD3 ganglioside and binds to immature neuroectodermal cells and to developing oligodendrocytes in forebrain and cerebellum; and (3) an antibody to the intermediate filament, vimentin. With time, two different populations emerged, both of which seemed to be derivatives of radial cells. One cell type expressed the astrocyte intermediate filament, GFAP, in addition to vimentin. GFAP‐containing cells eventually took on the forms of astrocytes in gray and white matter. The other type expressed carbonic anhydrase, an enzyme characteristic of oligodendrocytes and enriched in myelin. Carbonic anhydrase‐positive cells eventually developed into small cells with oligodendrocyte morphology. Our observations suggest a common lineage for astrocytes and oligodendrocytes from radial cells during spinal cord gliogene

ISSN:0360-4012    

DOI:10.1002/jnr.490210208

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY

摘要:

AbstractWe have used the monoclonal antibody A2B5(which binds to subclasses of surface gangliosides) to select glial precursor cells from postnatal rat brain and compare their properties in culture with those of the bipotential O‐2A progenitor cells of newborn optic nerve. Two methods, fluorescence‐activated cell sorting (FACS) and differential adhesion, resulted in>90% enrichment in A2B5‐positive bipolar cells and multipolar cells with short processes. These cells expressed vimentin and reacted with yet another antibody (NSP4), which binds to O‐2A progenitor cells of optic nerve. The 2–10% of the remaining cells consisted of type 1 astrocytes and/or microglial cells. When maintained in defined medium for 3 days, 28‐40% of A2B5‐positive cells incorporated thymidine, while most other cells became differentiated into galactocerebroside‐positive oligodendrocytes. In the presence of 10% fetal calf serum for 3 days, over 50% of the cells developed a stellate phenotype and expressed GFAP, characteristic of type 2 astrocytes. This phenotypic plasticity of the A2B5positive cells was also observed in clones derived from single cells grown on a layer of type 1 astrocytes. Thus, A2B5‐positive cells from cerebrum are O‐2A progenitors that can generate O‐2A lineage cells. The effects of the two growth factors, insulin and platelet derived growth factor (PDGF) (which is synthesized by type 1 astrocytes), were tested on cerebrum O‐2A progenitors. PDGF induced a doubling of the percentage of A2B5‐positive cells incorporating thymidine during a 20‐hr pulse and a large increase (up to 40‐fold) of the progenitor population over 3 days. The largest number of O‐2A lineage cells was obtained when purified progenitors were grown in the presence of PDGF and insulin. Thus, A2B5‐positive glial cells from cerebrum overall behave as the O‐2A progenitors of optic nerve, but they more readily divide than differentiate, as if they were at an earlier s

ISSN:0360-4012    

DOI:10.1002/jnr.490210209

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY

摘要:

AbstractThe iron transport glycoprotein, transferrin (Tf), localizes exclusively in oligodendrocytes in brain tissue sections. Previously, we showed that Tf is also expressed in oligodendrocytes in primary cultures established from newborn rat brains. Its developmental appearance precedes that of galactocerebroside (GC). In this study, Tf expression in primary brain cell cultures was investigated over a 4‐week period in relation to GC and myelin basic protein (MBP), respectively, early and late markers of oligodendrocyte development. From 9 days in vitro and thereafter, all Tf+cells were also found to be GC+. With increasing age the number of Tf+cells decreased while the number of MBP+cells increased. However, less than 10% of oligodendrocytes co‐expressed Tf and MBP at any age. MBP+cells were largely found in cell clusters which increased in size and number with age in culture. Interestingly, Tf+cells were located around the clusters of MBP+cells which displayed elaborate branched processes. The transient expression of Tf in oligodendrocytes which become MBP+, suggests a role for Tf in the early stages of myelinogenesis. The results also demonstrate the existence of three phenotypically distinct populations of oligodendrocytes. A new model of developmental and functional subpopulations of oligodendrocytes is propo

ISSN:0360-4012    

DOI:10.1002/jnr.490210210

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY

摘要:

AbstractThe aim of the present study was to prepare cultures enriched in type‐2 astrocytes (AS) and to analyze some of the properties of these cells over relatively long culture periods. Cultures enriched in type‐2 AS were obtained by subculturing, at low cell density and in the presence of fetal calf serum, a cell population containing numerous bipotential glial precursors. This cell population was detached mechanically from 2‐ to 3‐week primary mixed glial cultures prepared from 1‐day postnatal rat cerebral cortex. The cellular composition of the subcultures was analyzed immunocytochemically over a period of 3 weeks using various combinations of antibodies, recognizing a set of differentiated and a set of undifferentiated glial antigens (glial fibrillary acidic protein [GFAP], galactocerebroside, sulfatide, gangliosides binding the monoclonal antibodies A2B5 and LB1, fibronectin). Most LB1+, A2B5+glial precursors differentiated into type‐2 AS within a week. At this stage, type‐2 AS accounted for more than 70% of cells in the cultures and exhibited the characteristic features previously described for these cells (stellate shape, GFAP, LB1 and A2B5 positivity, ability to accumulate [3H]GABA and to synthesize chondroitin sulfate, low proliferative activity). About one third of the type‐2 AS also were recognized by O4 (antisulfatide) antibodies. The major contaminants were macrophages (10–15%) and fibroblastic cells (5–10%). In longer term cultures, type‐2 AS tended to lose several of these features. Many acquired a flat, polygonal shape and lost LB1 positivity. The ability to accumulate [3H]GABA progressively decreased, as did the expression of chondroitin sulfate, although to a lesser degree. Although losing several of their properties, type‐2 AS did not appear to acquire the properties of type‐1 AS: their proliferative activity remained very low, and they did not express class II antigens of the major histocompatibility complex upon stimulation with γ‐interferon. Some bec

ISSN:0360-4012    

DOI:10.1002/jnr.490210211

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY

摘要:

AbstractWe investigated the mechanisms by which insulin‐like growth factor I (IGF‐I) acts to increase the number of oligodendrocytes that develop in cultures of cells explanted from perinatal rat cerebrum. Fluorescence‐activated cell sorting was used to isolate bipotential A2B5‐positive oligodendrocyte‐type 2 astrocyte (O‐2A) progenitor cells, which were then inoculated as single cells into microculture wells containing feeder layers of X‐irradiated type 1 astrocytes. Addition of 100 ng/ml IGF‐I to the culture medium increased the growth rate and the ultimate size reached by the resulting clones during the 18‐day experimental period. Moreover, 75–80% of the cells in the IGF‐I‐treated clones differentiated into galactocerebroside (GC)‐positive oligodendrocytes, whereas only 25–30% became oligodendrocytes in the absence of IGF‐I. IGF‐I did not increase the number of type 2 astrocytes that developed in the clones. IGF‐I appeared to have the greatest effect on growth and differentiation at a stage when the majority of the cells in the clones were at an intermediate stage of development, characterized by the expression of A2B5 and O4 glycolipid antigens but not GC. Analysis of the effects of IGF‐I on O4‐positive, GC‐negative intermediate precursor cells revealed a two to fivefold increase in the number of cells that incorporated3H‐thymidine into their DNA during a 5‐h pulse. Moreover, IGF‐I increased the number of cell sorter‐purified O4‐positive cells that developed into oligodendrocytes 4–8 days later. Therefore, IGF‐I acts in two different ways to promote oligodendrocyte development: It promotes proliferation of precursor cells in the O‐2A lineage, and it induces precurso

ISSN:0360-4012    

DOI:10.1002/jnr.490210212

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY