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Myelin-associated glycoprotein immunoreactive material: An early neuronal marker of dorsal root ganglion cells during chick development
Abstract
Immunostaining of myelin-associated glycoprotein (MAG) was performed in chick dorsal root ganglia (DRG) during development. The MAG-immunoreactive material appeared first around 7 days of incubation in immature neurons of DRG. Immunoprecipitates first confined to one pole of nucleus were gradually redistributed in the perinuclear Golgi apparatus of small DRG cells. Thus MAG may be used in the chick embryo as an early marker of primary sensory neurons of class B.
... For decades, the two neuron populations of the chick embryo DRG were designated LV and MD cells, and the issue of homology with other adult vertebrate DRG cells did not arise. Recently, however, (Barakat et al. 1985;Philippe et al. 1986) have applied the A-and B-neuron terminology to the chick embryo cells, presumably because myelin-associated glycoprotein immunoreactivity was localized specifically in chick embryo MD cells (Omlin et al. 1984), and this immunocytochemical stain-Ing In hatching-age chick ganglion cells was correlated with B-neuron morphology (Philippe et al. 1986). Other grounds for postulating an equivalence between B-neurons and MD cells are: (1) Both MD neurons (Hamburger & Levi-Montalcini 1949) and B-neurons (Lawson 1979, mouse;Lawson & Biscoe 1979, mouse) are born in the second of the two overlapping waves of cell proliferation. ...
... For decades, the two neuron populations of the chick embryo DRG were designated LV and MD cells, and the issue of homology with other adult vertebrate DRG cells did not arise. Recently, however, (Barakat et al. 1985;Philippe et al. 1986) have applied the A-and B-neuron terminology to the chick embryo cells, presumably because myelin-associated glycoprotein immunoreactivity was localized specifically in chick embryo MD cells (Omlin et al. 1984), and this immunocytochemical stain-Ing In hatching-age chick ganglion cells was correlated with B-neuron morphology (Philippe et al. 1986). Other grounds for postulating an equivalence between B-neurons and MD cells are: (1) Both MD neurons (Hamburger & Levi-Montalcini 1949) and B-neurons (Lawson 1979, mouse;Lawson & Biscoe 1979, mouse) are born in the second of the two overlapping waves of cell proliferation. ...
... Expression of MKI67 [28] and PCNA [29] genes, two well-established cell proliferation markers, showed the highest correlations with kinases strongly associating to mitosis and cell cycle. Similarly, LDHC (germ-cell specific marker) [30], PTPRC (marker for hematopoiesis) [31], VCAM1 (endothelial/vascular cell marker) [32], KRT19 (epithelial marker) [33], MAG (neuronal cell marker) [34] and CAV3 (myocyte marker) [35] correlated with the kinase genes with corresponding functional associations. ...
Kinases play key roles in cell signaling and represent major targets for drug development, but the regulation of their activation and their associations with health and disease have not been systematically analyzed. Here, we carried out a bioinformatic analysis of the expression levels of 459 human kinase genes in 5681 samples consisting of 44 healthy and 55 malignant human tissues. Defining the tissues where the kinase genes were transcriptionally active led to a functional genomic taxonomy of the kinome and a classification of human tissues and disease types based on the similarity of their kinome gene expression. The co-expression network around each of the kinase genes was defined in order to determine the functional context, i.e. the biological processes that were active in the cells and tissues where the kinase gene was expressed. Strong associations for individual kinases were found for mitosis (69 genes, including AURKA and BUB1), cell cycle control (73 genes, including PLK1 and AURKB), DNA repair (49 genes, including CHEK1 and ATR), immune response (72 genes, including MATK), neuronal (131 genes, including PRKCE) and muscular (72 genes, including MYLK2) functions. We then analyzed which kinase genes gain or lose transcriptional activity in the development of prostate and lung cancers and elucidated the functional associations of individual cancer associated kinase genes. In summary, we report here a systematic classification of kinases based on the bioinformatic analysis of their expression in human tissues and diseases, as well as grouping of tissues and tumor types according to the similarity of their kinome transcription.
... This observation fits well with the known oncodevelopmental nature of PLAP, with ectopic expression being common in various types of cancers, with uterine and ovarian cancers being particularly well defined as PLAP-positive [31,32]. Finally, MAG, a neuronal cell marker [33] , showed the highest expression in central nervous system, and to a lesser extent in gliomas (Figure 3c), again a GeneSapiens profile that could be expected for this well-known marker gene. Additional examples are given in Additional data files 4 and 5, and dozens of known tissue-specific genes or biomarkers can be evaluated through the online tool for exploring tissue-and disease-specific gene expression patterns. ...
Our knowledge on tissue- and disease-specific functions of human genes is rather limited and highly context-specific. Here, we have developed a method for the comparison of mRNA expression levels of most human genes across 9,783 Affymetrix gene expression array experiments representing 43 normal human tissue types, 68 cancer types, and 64 other diseases. This database of gene expression patterns in normal human tissues and pathological conditions covers 113 million datapoints and is available from the GeneSapiens website.
... The sensory neurons identified by galNac-binding lectins correspond in size and location to the population of DRG neurons that express SP, CGRP (New and Mudge, 1986; Du et al., 1987), myelin-associated glycoprotein (MAG) (Omlin et al., 1985), and the lactoseries glycoconjugates AC4 antigen and SSEA-1 (Scott et al., 1987 ). The extent to which these markers coexist in individual neurons is unknown, The glycoconjugate described here appears much later in development than SP (New and Mudge, 1986; Du et al., 1987) MAG (Philippe et al., 1986), or lactoseries glycoconjugates (Scott et al., 1987 ). Thus, its expression is likely to be regulated differently , and it undoubtedly serves a different function from the earlier-appearing glycoconjugates and peptides. ...
We screened a variety of lectins with different sugar specificates to determine whether subpopulations of dorsal root ganglion (DRG) neurons in the chick can be distinguished by the carbohydrates they express. Of the 15 lectins tested only those that recognize N-acetylgalactosamine (galNac) residues labeled a subset of DRG neurons. For example, Dolichos biflorus (DBA) labeled a population of small-diameter neurons in the dorsomedial DRG and their terminals in the dorsal horn in hatchling chicks. Staining of live neurons in vitro demonstrated that DBA was binding to the cell surface. Labeling first appeared in sensory neurons at about St.38 (E12) and in dorsal horn laminae 1 and 2 at about St.42 (E16). Fainter labeling appeared somewhat later in lamina 3, after hatching. Labeling of the tissue sections was eliminated by chloroform: methanol extraction and reduced by alpha-N-acetylgalactosaminidase digestion, but survived trypsinization. Together these results suggest that a subset of DRG neurons in the chick can be identified by the presence of a cell surface glycoconjugate, perhaps a glycolipid, containing terminal alpha-linked galNac residues.
... We feel that the present study gives the first unequivocal evidence of the absence of MAG in compact myelin and thus settles a controversy. Also, in contrast to a previous immunocytochemical study in the peripheral nervous system of the chicken, we have not found detectable levels of MAG on neurons (Omlin et al., 1985; Philippe et al., 1986). Although functional relationships in cell interactions cannot be deduced from morphological data, our observations suggest that LI and N-CAM are involved in axon fasciculation as it has been observed previously in the central nervous system (Fischer et al., 1986) and mediate initial contacts between axons and Schwann cells. ...
The cellular and subcellular localization of the neural cell adhesion molecules L1, N-CAM, and myelin-associated glycoprotein (MAG), their shared carbohydrate epitope L2/HNK-1, and the myelin basic protein (MBP) were studied by pre- and post-embedding immunoelectron microscopic labeling procedures in developing mouse sciatic nerve. L1 and N-CAM showed a similar staining pattern. Both were localized on small, non-myelinated, fasciculating axons and axons ensheathed by non-myelinating Schwann cells. Schwann cells were also positive for L1 and N-CAM in their non-myelinating state and at the onset of myelination, when the Schwann cell processes had turned approximately 1.5 loops. Thereafter, neither axon nor Schwann cell could be detected to express the L1 antigen, whereas N-CAM was found in the periaxonal area and, more weakly, in compact myelin of myelinated fibers. Compact myelin, Schmidt-Lanterman incisures, paranodal loops, and finger-like processes of Schwann cells at nodes of Ranvier were L1-negative. At the nodes of Ranvier, the axolemma was also always L1- and N-CAM-negative. The L2/HNK-1 carbohydrate epitope coincided in its cellular and subcellular localization most closely to that observed for L1. MAG appeared on Schwann cells at the time L1 expression ceased. MAG was then expressed at sites of axon-myelinating Schwann cell apposition and non-compacted loops of developing myelin. When compaction of myelin occurred, MAG remained present only at the axon-Schwann cell interface; Schmidt-Lanterman incisures, inner and outer mesaxons, and paranodal loops, but not at finger-like processes of Schwann cells at nodes of Ranvier or compacted myelin. All three adhesion molecules and the L2/HNK-1 epitope could be detected in a non-uniform staining pattern in basement membrane of Schwann cells and collagen fibrils of the endoneurium. MBP was detectable in compacted myelin, but not in Schmidt-Lanterman incisures, inner and outer mesaxon, paranodal loops, and finger-like processes at nodes of Ranvier, nor in the periaxonal regions of myelinated fibers, thus showing a complementary distribution to MAG. These studies show that axon-Schwann cell interactions are characterized by the sequential appearance of cell adhesion molecules and MBP apparently coordinated in time and space. From this sequence it may be deduced that L1 and N-CAM are involved in fasciculation, initial axon-Schwann cell interaction, and onset of myelination, with MAG to follow and MBP to appear only in compacted myelin. In contrast to L1, N-CAM may be further involved in the maintenance of compact myelin and axon-myelin apposition of larger diameter axons.
... The sensory neurons that give rise to the primary afferent projection to the spinal cord constitute a functionally diverse population. They have been categorized variously according to peripheral target tissue (cutaneous, muscle, visceral), target sensory organ (Pacinian corpuscle, hair follicle, muscle spindle, tendon organ, etc.), axonal conduction velocity and caliber (type Ia, Ib, II, etc.), sensory modality (stretch, temperature, pain, etc.), neurotransmitter profile (glutamate, substance P, CGRP), soma size and localization (Hamburger and Levi-Montalcini 1949;Hamburger et al. 1981), cytoplasmic antigens (Omlin et al. 1985;Philippe et al. 1986), neurotrophin dependence (LoPresti and Scott 1994;Snider 1994;Bothwell 1995), and the composition of cell surface molecules (Tanaka and Obata 1984;Dodd et al. 1984Dodd et al. , 1988Jessell 1985, 1986;Jessell and Dodd 1985;Mori 1986;Nakagawa et al. 1986;Scott et al. 1990). A great deal of effort has been directed towards defining factors that determine sensory neuron phenotypes (Marusich et al. 1986;New and Mudge 1986;Rohrer et al. 1986;Rohrer and Thoenen 1987;Ernsberger and Rohrer 1988;Marusich and Weston 1988;Sieber-Blum 1991;Anderson 1993Anderson , 1994Vogel et al. 1993;Davies 1994;Lewin and Barde 1996), elucidating the central connections specific to the different categories of sensory neurons (Brown et al. 1977(Brown et al. , 1978Brown and Fyffe 1978, 1981Burke et al. 1979;Ishizuka et al. 1979;Light and Perl 1979;Brown 1981;Jhaveri and Frank 1983;McMahon and Wall 1985;Sugiura et al. 1989), and understanding how these connections are established during development (Saito 1979;Eide et al. 1982;Smith 1983;Kudo and Yamada 1987;Lee et al. 1988; Davis et al. 1989;Mendelson et al. 1992;Snider et al. 1992;Frank and Wenner 1993;Seebach and Ziskind-Conhaim 1994;Zhang et al. 1994;Eide and Glover 1995;Mirnics and Koerber 1995;Wenner and Frank 1995;Messersmith et al. 1995;). ...
The central projections of specific subpopulations of lumbar primary afferents were selectively labeled with the lipophilic tracer DiI in fixed preparations of the chicken embryo. Muscle or cutaneous afferents were selectively labeled by applying DiI to identified peripheral nerves. Medial or lateral afferent populations were selectively labeled by partially lesioning the dorsal root. Muscle and cutaneous afferent populations each contribute to both the medial and the lateral afferent populations. Medial muscle afferents terminate in the intermediate zone and lateral motor column proximally, but only in the intermediate zone distally. Lateral muscle afferents terminate in a ventrolateral region of the dorsal horn both proximally and distally. Medial cutaneous afferents terminate predominantly in lamina III, but a few terminate in the medial region of the intermediate zone. Lateral cutaneous afferents terminate in lamina II and in a ventrolateral region of the dorsal horn. On the basis of the principle termination patterns, specific termination fields were defined and related to the classical cytoarchitectonics of the spinal gray matter. Differential retrograde tracing from the spinal cord with fluorescent dextran-amines demonstrated that the medial afferents originate from the earlier-generated ventrolateral population of large sensory cell bodies, while the lateral afferents originate from the later-generated dorsomedial population of small sensory cell bodies. The medial afferents establish their central projections earlier than the lateral afferents, but for each subpopulation the initial pattern of termination prefigures the mature pattern, throughout the segmental range of the collaterals. Birthdating with 3H-thymidine showed that potential target neurons in the different terminal fields within the dorsal horn are born at different times. In particular, interneurons in lamina II are born after those in lamina III, paralleling the early and late termination of cutaneous afferents in these laminae. Our observations support the notion that primary afferents recognize specific cues in the spinal cord, but also implicate the relative timing of afferent and target differentiation as an important determinant of primary afferent termination patterns.
The primary sensory neurons enclosed in the dorsal root ganglion (DRG) offer neurobiologists the possibility to test the influence exerted by environmental conditions on the phenotypes expressed by DRG cells. Although the population of the primary sensory neurons originates from a common source, the neural crest, DRG cells differentiate into various neuronal subpopulations which may be distinguished by their peripheral and central projections, electrophysiological properties, enzymatic equipment, pharmacological receptors, neurotransmitter content, cell surface antigens as well as cytochemical and ultrastructural characteristics. The proportion of these neuronal subpopulations shows great fluctuations with the age (Droz and Kazimierczak, 1987), position of the DRG along the neuraxis (Masurich et al., 1986a), or alteration of the innervated targets (Carr and Simpson, 1978; Philippe et al., 1988). It is therefore important to specify the cellular mechanisms which are involved in the remodeling of the neuronal population in DRG.
The peripheral nervous system (PNS) has classically been separated into a somatic division composed of both afferent and efferent pathways and an autonomic division containing only efferents. J. N. Langley, who codified this asymmetrical plan at the beginning of the twentieth century, considered different afferents, including visceral ones, as candidates for inclusion in his concept of the “autonomic nervous system” (ANS), but he finally excluded all candidates for lack of any distinguishing histological markers. Langley's classification has been enormously influential in shaping modern ideas about both the structure and the function of the PNS. We survey recent information about the PNS and argue that many of the sensory neurons designated as “visceral” and “somatic” are in fact part of a histologically distinct group of afferents concerned primarily autonomic function. These afferents have traditionally been known as “small dark” neurons or B-neurons. In this target article we outline an association between autonomic and B-neurons based on ontogeny, cell phenotype, and functional relations, grouping them together as part of a common reflex system involved in homeostasis. This more parsimonious classification of the PNS, made possible by the identification of a group of afferents associated primarily with the ANS, avoids a number of confusions produced by the classical orientation. It may also have practical implications for an understanding of nociception, homeostatic reflexes, and the evolution of the nervous system.
During embryonic development, skin sensory innervation in the chick hindlimb is established in a precise and orderly fashion. From their earliest outgrowth, axons of each dorsal root ganglion (DRG) project through the limb via the correct pathways to establish their dermatome in the appropriate location. By making experimental manipulations in young embryos and mapping the resulting innervation patterns, we have begun to reveal some of the mechanisms involved. For example, when several DRG are eliminated, the axonal projections of intact DRG are shifted and the dermatomes are enlarged, suggesting that competition among axons is important in determining both the contribution of DRG to axonal pathways and the borders between dermatomes. Further, when the rostrocaudal order of DRG is reversed, skin sensory innervation patterns tend to mimic the motor projection patterns, implying that motor axons may guide the growth of sensory axons. However, neither of these mechanisms can accountfully for the precision with which skin sensory innervation patterns are established. Thus, it is likely that skin sensory neurons use multiple, redundant developmental cues in selecting their appropriate pathways and targets.
1.1. A variety of colloidal gold-labelled lectins with different sugar specificities to determine whether different nerve and glial cells of the snail Helix pomatia cultured in vitro, can be distinguished by the carbohydrates that they express was screened. The analysis of lectin binding has shown substantial differences in the carbohydrate pattern between nerve and glial cells and between the soma of monoaminergic and peptidergic neurons.2.2. The surface of monoaminergic and peptidergic neurons contains N-acetylglucosamine and N-acetyllactosamine determinants, and does not exhibit neuraminic acid and complex branched N-glycosyl chains. Moreover, N-acetylgalactosamine can be detected on peptidergic neuron membranes only.3.3. N-Acetylglucosamine residues are not present on the surface of the glial cells, and the density of the N-acetyllactosamine and/or terminal β-galactose residues is much higher here than on the surface of the nerve cells.4.4. These results suggest that nerve cells in the snail brain can be distinguished from glial cells by the presence of a cell-surface glycoconjugate containing terminal N-acetyl-d-glucosamine residues, whereas peptidergic neurons can be distinguished from monoaminergic neurons by the presence of a surface glycoconjugate containing terminal α-linked N-acetyl-d-galactosamine residues.
The expression of substance P (SP) was studied in sensory neurons of developing chick lumbosacral dorsal root ganglia (DRG) by using a mixture of periodic acid, lysine and paraformaldehyde as fixative and a monoclonal antibody for SP-like immunostaining. The first SP-like-immunoreactive DRG cells appeared first at E5, then rapidly increased in number to reach a peak (88% of ganglion cells) at E8, and finally declined (59% at E12, 51% after hatching). The fall of the SP-like-positive DRG cells resulted from two concomitant events affecting a subset of small B-neurons: a loss of neuronal SP-like immunoreactivity and cell death. After one hindlimb resection at an early (E6) or late (E12) stage of development (that is before or after establishment of peripheral connections), the DRG were examined 6 days later. In both cases, a drastic neuronal death occurred in the ispilateral DRG. However, the resection at E6 did not change the percentage of SP-like-positive neurons, while the resection at E12 severely reduced the proportion of SP-like-immunoreactive DRG cells (25%). In conclusion, connections established between DRG and peripheral target tissues not only promote the survival of sensory neurons, but also control the maintenance of SP-like-expression. Factors issued from innervated targets such as NGF would support the survival of SP-expressing DRG cells and enhance their SP content while other factors present in skeletal muscle or skin would hinder SP expression and therefore lower SP levels in a subset of primary sensory neurons.
During the ontogenesis of dorsal root ganglia (DRG), the immunoreactivity to substance P (SP) and calbindin D-28k (CaBP) appears in chickens at embryonic day 5 (E5) and E10 respectively. To establish the birthdates of primary sensory neurons expressing SP or CaBP, chick embryos were given repetitive intra-amniotic injections of [3H]-thymidine. The neuroblasts giving rise to SP-expressing neurons were labeled up to E6 while those generating CaBP-immunoreactive neurons stopped to incorporate [3H]-thymidine before E5.5. This finding indicates that neurons exhibiting distinct phenotypes may originate from neuroblasts which arrest to proliferate at close but distinct stages of development. To determine whether SP and CaBP are co-expressed or not in DRG neurons, chick embryos at E12, E18, and chickens two weeks after hatching were perfused and fixed to detect simultaneously SP- and CaBP-immunoreactivity in DRG sections. The results showed that SP and CaBP were transiently co-expressed by a subset of neurons at E12. Later, however, the SP-immunoreactivity was gradually lost by these ganglion cells, so that the SP- and CaBP-immunoreaction defined two distinct neuronal subpopulations after hatching. In conclusion, most CaBP-immunoreactive DRG cells derive from a subset of neurons in which SP and CaBP are transiently co-localized.
We have previously isolated and sequenced the chicken nerve growth factor (NGF) gene and now, from the deduced amino acid sequence, selected and produced peptides suitable for use as antigens. Anti-sera raised against these peptides inhibit the biological activity of a partially purified preparation of native chicken NGF and, when used in immunocytochemical studies, allow the visualisation of sensory neurons accumulating endogenous NGF. Immunoreactive cells form a distinct population of small neurons which may correspond to the well-described neurons generated in the dorsomedial area of spinal ganglia. We conclude that two subpopulations of neurons exist within dorsal root ganglia, whose requirements for, and use of, NGF may be quite distinct.
Counts performed on dissociated cell cultures of E10 chick embryo dorsal root ganglia (DRG) showed after 4-6 days of culture a pronounced decline of the neuronal population in neuron-enriched cultures and a net gain in the number of ganglion cells in mixed DRG cell cultures (containing both neurons and nonneuronal cells). In the latter case, the increase in the number of neurons was found to depend on NGF and to average 119% in defined medium or 129% in horse serum-supplemented medium after 6 days of culture. The lack of [3H]thymidine incorporation into the neuronal population indicated that the newly formed ganglion cells were not generated by proliferation. On the contrary, the differentiation of postmitotic neuroblasts present in the nonneuronal cell compartment was supported by sequential microphotographs of selected fields taken every hour for 48-55 hr after 3 days of culture. Apparently nonneuronal flat dark cells exhibited morphological changes and gradually evolved into neuronal ovoid and refringent cell bodies with expanding neurites. The ultrastructural organization of these evolving cells corresponded to that of primitive or intermediate neuroblasts. The neuronal nature of these rounding up cell bodies was indeed confirmed by the progressive expression of various neuronal cell markers (150 and 200-kDa neurofilament triplets, neuron specific enolase, and D2/N-CAM). Besides a constant lack of immunoreactivity for tyrosine hydroxylase, somatostatin, parvalbumin, and calbindin-D 28K and a lack of cytoenzymatic activity for carbonic anhydrase, all the newly produced neurons expressed three main phenotypic characteristics: a small cell body, a strong immunoreactivity to MAG, and substance P. Hence, ganglion cells newly differentiated in culture would meet characteristics ascribed to small B sensory neurons and more specifically to a subpopulation of ganglion cells containing substance P-immunoreactive material.
Neuronal precursor cells present in dorsal root ganglia (DRG) during early development have been previously shown to differentiate in vitro to neurons, as characterized by morphology, cell surface antigens, and electrophysiological properties (H. Rohrer, S. Henke-Fahle, T. El-Sharkawy, H. D. Lux, and H. Thoenen, 1985, Embo J. 4, 1709-1714). In the present study the conditions necessary for the initial differentiation and long-term survival of these cells were established, and the neurotransmitter phenotype of the newly differentiated neurons was analyzed. Neuronal precursor cells isolated from chick DRG at Embryonic Day 6 (E6) were found to require the presence of a polyornithine substrate coated with either laminin or fibronectin for initial neurite production and long-term survival. Neurons were unable to develop on polyornithine alone or on polyornithine coated with BSA. The survival and neurite outgrowth from neuronal precursor cells was not affected by the presence of nerve growth factor (NGF) during the first 9 hr in culture. NGF also had no effect on the proportion of cells expressing the neuron-specific Q211 antigen. However, after this initial differentiation period the neurons did require the presence of a survival factor. The neurons could be maintained for at least 6 days in culture both in the presence of NGF and in the presence of brain-derived neurotrophic factor (BDNF). At saturating concentrations of both survival factors no additive effects could be observed, indicating a complete overlap of NGF- and BDNF-responsiveness. Although the same proportion of cells survived with either NGF or BDNF during the first 3 days in culture, survival decreased in the presence of BDNF but not in the presence of NGF during the following 3 days in culture. The loss of BDNF responsiveness in vitro was also observed in vivo. After 6 days in culture about 70% of the neurons expressed substance P immunoreactivity, and approximately the same proportion was positive for myelin-associated glycoprotein immunoreactivity. The neurons did not express properties of adrenergic neurons such as tyrosine hydroxylase immunoreactivity or norepinephrine uptake. These findings indicate that the neuronal precursor cells from E6 DRG acquire the same characteristics in vitro as in their normal in vivo environment.
The aim of this study was to investigate the influence of collagen or polyornithine substrates on cell migration in explant cultures of dorsal root ganglia (DRG) by means of light microscopy and immunocytochemistry. Myelin-associated glycoprotein (MAG) immunoreactivity was used to characterize the subpopulation of small B ganglion cells, whereas neuron-specific enolase (NSE) immunoreactivity acted as a general neuronal cell marker. After a few days in culture, DRG explants grown on collagen substrate showed a flattened shape consisting of a core surrounded by a crown of neurites, which were mixed up with migrating cells of different types. These migrating cells were immunostained for both MAG and NSE and were observed after 7 days in the vicinity of the explant core, then after 14 days also at a distance from the explant core. In contrast, even after 14 days in culture, explants grown on polyornithine substrate maintained a globular shape. The MAG-positive ganglion cells were confined to the explant core and no cell migration was observed on this type of substrate. MAG immunoprecipitates located at the ganglion cell surface were observed in explants cultured on polyornithine, but rarely on collagen substrate. In conclusion, it is suggested that this pattern of intracellular distribution of MAG immunoreactive material could reflect interactions between cell surface and extracellular matrix, and could condition the migratory ability of small ganglion cells.
The protein encoded by the rat brain cDNA 1B236 has been shown to be identical to myelin-associated glycoprotein (MAG). In this report we describe the cellular distribution of 1B236/MAG mRNA transcripts in rat brain by using in situ hybridization. At postnatal day 20, large numbers of 1B236/MAG mRNA-containing oligodendrocytes are concentrated in myelinated fiber tracts and throughout gray matter regions. The presence of high levels of 1B236/MAG mRNA within oligodendrocytes at postnatal day 20 is consistent with the proposed role of MAG in formation of the myelin sheath during development. In the adult brain, our results suggest that not only is 1B236/MAG mRNA expressed at reduced levels within oligodendrocytes but also 1B236/MAG or a 1B236/MAG-like mRNA is present within neurons. This localization is consistent with the results of previous immunocytochemical studies using antibodies against the 1B236/MAG mRNA with different cell-type-specific patterns of expression suggests that oligodendrocytes and neurons employ different mechanisms for regulating the same gene. Thus, different cell types may use a similar cell adhesion molecule both during myelinogenesis and in the mature nervous system.
The localization of the cell adhesion molecules L1, neural cell adhesion molecule (N-CAM), and myelin-associated glycoprotein (MAG) was studied immunohistologically at the light and electron microscopic levels and immunochemically in the developing and adult mouse optic nerve and retina. The neural adhesion molecule L1 is strongly expressed on the shafts of fasciculating unmyelinated axons at all ages studied from embryonic day 15 through adulthood. Growth cones of retinal ganglion cell axons were weakly L1-positive or L1-negative when contacting glial cells. Unmyelinated axons were not only L1-positive when contacting each other but also when contacting glia, whereas contacts between glial cells were L1-negative at all developmental stages. The same axons that expressed L1 in their fasciculating state in the unmyelinated retinal nerve fiber layer or in the unmyelinated optic nerve head became L1-negative when enwrapped by myelin in the optic nerve proper. At all stages of development N-CAM showed profuse labeling on fasciculating axons, growth cones, and their contact sites with glial cells as well as contacts between glial cells. In contrast to L1, axons remained N-CAM-positive when becoming myelinated. Sometimes, N-CAM was found in compact myelin. However, N-CAM was absent from glial surfaces contacting basement membranes at the interface to meninges, blood vessels, and the vitreous body of the eye. MAG was first detectable intracellularly in oligodendrocytes associated with the endoplasmic reticulum and Golgi apparatus before it became apparent at the cell surface. There it was present on oligodendrocytes prior and during the first stages of ensheathment of axons, both on cell body and processes. After formation of compact myelin MAG remained strongly expressed periaxonally and was only weakly detectable in noncompacted myelin including inner mesaxon and paranodal loops. None of the adhesion molecules was detectable on extracellular matrix, in the meninges, or on endothelial cells. Immunochemical analysis of antigen expression at different developmental stages was in agreement with the immunohistological data.
We infer from these observations that L1 is involved in stabilization not only of axon-axon, but also axon-glia contacts, while the more dynamic structure of the growth cone generally expresses less L1. A differential expression of L1 along the course of an axon—being present on its unmyelinated, but absent on its myelinated part—further supports the notion that L1 may be involved in the stabilization of axonal fascicles but not of axon-myelin contacts. Since axon-astrocyte appositions are L1-negative at the node of Ranvier and L1-positive in nonmyelinated areas, L1 appears to play a regionally differential functional role in neuron-astrocyte interactions. MAG seems to be involved in the initiation of axon-oligodendrocyte interactions before the onset of my-elination and in the stabilization of neuron-oligodendrocyte and oligodendro- cyte-oligodendrocyte contacts in the mature myelin. Because of its general occurrence at contacts between all cell types studied except for the astroglial- basement membrane apposition, N-CAM appears to be responsible for the general stabilization of tissue integrity at all developmental stages studied and in the adult.
Investigation of the early phases of the development of primary sensory neurons has been limited to cells obtained from sensory ganglia. Due to the lack of an early, lineage-specific marker for sensory neuroblasts, it has not been possible to use the neural crest, which gives rise to all spinal and some cranial primary sensory neurons, as a source of precursor cells. In the present study, we show that in neural crest derivatives of the quail embryo, the stage-specific embryonic antigen-1 (SSEA-1) is expressed specifically by developing sensory neuroblasts. The monoclonal antibodies anti-SSEA-1 and AC4 were used to characterize sensory neuron development in vivo and in neural crest cell cultures. In the rat and mouse, both antibodies recognize the same carbohydrate sequence [galactose beta 1-4(fucose alpha 1-3)N-acetylglucosamine] which characterizes SSEA-1. In the quail embryo, this epitope is a marker with several attractive characteristics. Among neural crest derivatives, it is specific for the sensory lineage and is expressed by all detectable sensory neuroblasts at all spinal axial levels. In addition, the carbohydrate sequence appears early and persists throughout development. Expression of SSEA-1 was also studied in neural crest cell cultures, in which two populations of sensory neuroblasts were observed. One population differentiated before or shortly after explanation into culture; these cells did not emigrate from the neural tube. A second population appeared in older cultures. Forming the leading edge of the emigrating neural crest cells, they became SSEA-1+ 3 days after the nonmigrating SSEA-1+ cells. Double staining experiments revealed no obvious differences between the two populations with regard to morphology, neurofilament expression, and neurotransmitter content.
Antibodies to a variety of carbohydrate differentiation antigens were screened in dorsal root ganglia (DRG) of hatchling chicks with the goal of identifying markers for subpopulations of sensory neurons. Two antibodies, AC4 and anti-SSEA-1, which recognize a lactoseries glycoconjugate(s), labeled a subset of small and medium diameter neurons in the dorsomedial DRG. This antigen was present early in embryonic development, being expressed on sensory neuron precursors prior to their withdrawal from the cell cycle, and appeared to be associated with a cell-surface glycolipid. Immunoreactive neurons were supported in vitro primarily by brain-derived neurotrophic factor (BDNF), to a lesser extent by neurotrophin-3 (NT-3), but not by nerve growth factor (NGF). The subpopulation of sensory neurons identified by these antibodies is likely to be functionally diverse, having targets in both skin and muscle.
In a previous study, we have demonstrated that an ovalbumin-like antigen is present within approximately one-half of all neurons of chicken spinal ganglia. The current study demonstrates this antigen co-localizes absolutely with neural intermediate filament protein (Peripherin) in small to medium-sized neurons of spinal ganglia. While the function of ovalbumin in neurons is unknown, its precise co-localization with Peripherin suggests a functional role restricted to neurons of a defined phenotype.
Olig2, a basic helix-loop-helix (bHLH) transcription factor, is expressed in a restricted domain of the spinal cord ventricular zone that sequentially generates motoneurons and oligodendrocytes. Just prior to oligo-dendrocyte precursor formation, the domains of Olig2 and Nkx2.2 expression switch from being mutually exclusive to overlapping, and Neurogenins1 and 2 are extinguished within this region. Coexpression of Olig2 with Nkx2.2 in the spinal cord promotes ectopic and precocious oligodendrocyte differentiation. Both proteins function as transcriptional repressors in this assay. This effect is blocked by forced expression of Neurogenin1. By contrast, misexpression of Olig2 alone derepresses Neurogenins and promotes motoneuron differentiation. Olig2 therefore functions sequentially in motoneuron and oligodendrocyte fate specification. This dual action is enabled by spatio-temporal changes in the expression domains of other transcription factors with which Olig2 functionally interacts.
Brain spectrin, a membrane-related cytoskeletal protein, exists as two isoforms. Brain spectrin 240/235 is localized preferentially in the perikaryon and axon of neuronal cells and brain spectrin 240/235E is found essentially in the neuronal soma and dendrites and in glia (Riederer et al., 1986, J. Cell Biol., 102, 2088 - 2097). The sensory neurons in dorsal root ganglia, devoid of any dendrites, make a good tool to investigate such differential expression of spectrin isoforms. In this study expression and localization of both brain spectrin isoforms were analysed during early chicken dorsal root ganglia development in vivo and in culture. Both isoforms appeared at embryonic day 6. Brain spectrin 240/235 exhibited a transient increase during embryonic development and was first expressed in ventrolateral neurons. In ganglion cells in situ and in culture this spectrin type showed a somato - axonal distribution pattern. In contrast, brain spectrin 240/235E slightly increased between E6 and E15 and remained practically unchanged. It was localized mainly in smaller neurons of the mediodorsal area as punctate staining in the cytoplasm, was restricted exclusively to the ganglion cell perikarya and was absent from axons both in situ and in culture. This study suggests that brain spectrin 240/235 may contribute towards outgrowth, elongation and maintenance of axonal processes and that brain spectrin 240/235E seems to be exclusively involved in the stabilization of the cytoarchitecture of cell bodies in a selected population of ganglion cells.
In this chapter we shall be concerned with the interactions that occur between the dorsal root ganglia (DRGs) and their peripheral fields of innervation in the course of vertebrate development. The term “center”, as used here, refers to the neuronal cell bodies comprising the dorsal root ganglia. “Periphery” refers to the organ or field of innervation from which sensory nerve impulses originate.
Glycoproteins are well established as cell-surface components and appear to participate in specific cell-cell interactions. Myelin is formed as an extension of the plasma membrane of the oligodendrocyte in the central nervous system (CNS) and the Schwann cell in the peripheral nervous system (PNS). Therefore, it is reasonable to expect that glycoproteins could be present in the myelin membrane and be involved in the formation of the spiraled myelin sheath around the axon. Histochemical studies at the light-microscopic level by Wolman (1957) and Wolman and Hestrin-Lerner (1960) indicated the presence of polysaccharide in myelin. On the basis of early electron-microscopic studies, Robertson (1959) suggested that the intra-period region of peripheral myelin contained polysaccharide or glycoprotein material, possibly derived from the surface of the Schwann cell. Later, Peterson and Pease (1972) developed a specialized embedding technique and stained with silicotungstic acid to provide electron-microscopic evidence for the presence of glycoproteins in the intraperiod line of peripheral myelin, supporting Robertson’s earlier hypothesis. The first direct biochemical demonstration of glycoproteins in purified myelin was provided by Margolis (1967), who showed that N-acetylglucosamine is present in several protein fractions obtained from bovine CNS myelin and that glycosaminoglycans are not present. Nevertheless, the nature of the glycoprotein components in myelin remained poorly defined.
The unlabeled peroxidase-antiperoxidase method has been used with antiserum against "myelin-associated glycoprotein" to establish the presence of the glycoprotein in myelin and myelin-forming cells of the developing rat nervous system. Myelin-associated glycoprotein is found in oligodendroglial cytoplasm before the beginning of myelination. Staining intensity of oligodendroglia increases during early development and slowly declines during the period of rapid myelination. Myelin staining is confined to the periaxonal region of the myelin sheath and does not increase as large, compact sheaths are formed. Antiserum to central nervous system myelin-associated glycoprotein also stains Schwann cells in developing trigeminal ganglia and the periaxonal region of peripheral myelin sheaths.
FORTY-FIVE FIGURES The preparation of a series of normal stages of the chick embryo does not need justification at a time when chick ernbryos are not only widely used in descriptive and experimental embryology but are proving to be increasingly valuable in medical research, as in work on viruses and cancer. The present series was planned in connection with the preparation of a new edition of Lillie’s DeueZopmerzt of the Chick by the junior author. It is being published separately to make it accessible immediately to a large group of workers. Ever since Aristotle “discovered” the chick embryo as the ideal, object for embryological studies, the embryos have been described in terms of the length of time of incubation, and this arbitrary method is still in general use, except for the first three days of incubation during which more detailed characteristics such as the numbers of somites are applied. The shortcomings of a classification based on chronological age are obvious to every worker in this field, for enormous variations may occur in embryos even though all eggs in a setting are plmaced in the incubator at the same time. Many factors are responsible for the lack of correlation between chronological and structural age. Among these are : genetic differences in the rate of development of different breccls (eg., the embryo of the White Leghorn breed develops more 49
Biochemical and immunocytochemical investigations have shown that myelin-associated glycoprotein (MAG) is exclusively related
to myelin and myelin-forming cells in mammals. In the present study it was found that dorsal root ganglia in young chickens
display MAG-immunoreactive material in most small sensory neurons. The presence of MAG at the surface of small sensory neurons
raises the question of whether this glycoprotein acts as a cell adhesion molecule in lower vertebrates.
Antigen was identified histochemically without the use of labeled antibodies by the sequential application of (a) specific rabbit antiserum, (b) sheep antiserum to rabbit immunoglobulin G, (c) specifically purified, soluble horseradish peroxidase-anti-horseradish peroxidase complex (PAP), (d) 3,3'-diaminobenzidine and hydrogen peroxide and (e) osmium tetroxide. A simple method for preparation of high yields of PAP consisted of precipitation of antibody from specific rabbit antiserum with horseradish peroxidase (PO) at equivalence, solubilization of the washed precipitate with excess PO at pH 2.3, 1°C, followed by immediate neutralization and separation of PAP from PO by half-saturation with ammonium sulfate. The ratio of PO to anti-PO in PAP was 3:2 irrespective of the source of antiserum. PAP was heterogeneous on electrophoresis, homogeneous on sedimentation, diffusion and electron microscopy and consisted of pentagons with diameters of 205 Å. s20,w, 11.98 x 10–13; d20,w, 2.48 x 10–7; molecular weight by sedimentation velocity, 429,000, and equilibrium, 413,000. Sensitivity and specificity of immunohistochemical staining of spirochetes was about 100- to 1000-fold that of immunofluorescence. The unexpected ratio of PO to anti-PO is presumed to be due to stabilization by the pentagonal shape in which three corners are suspected to be PO and two antibody fragment Fc.
Phenotypes of sensory neurones in the chick dorsal root ganglia
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