Proliferation and migration of glial precursor cells in the developing rat spinal cord

The mechanisms that control the production and differentiation of glial cells during development are difficult to unravel because of displacement of precursor cells from their sites of origin to their permanent location. The two main neuroglial cells in the rat spinal cord are oligodendrocytes and astrocytes. Considerable evidence supports the view that oligodendrocytes in the spinal cord are derived from a region of the ventral ventricular zone (VZ). Some astrocytes, at least, may arise from radial glia. In this study a 5-Bromo-2′-deoxyuridine (BrdU) incorporation assay was used to identify proliferating cells and examine the location of proliferating glial precursor cells in the embryonic spinal cord at different times post BrdU incorporation. In this way the migration of proliferating cells into spinal cord white matter could be followed. At E14, most of the proliferating cells in the periventricular region were located dorsally and these cells were probably proliferating neuronal precursors. At E16 and E18, the majority of the proliferating cells in the periventricular region were located ventrally. In the white matter the number of proliferating cells increased as the animals increased in age and much of this proliferation occurred locally. BrdU labelling showed that glial precursor cells migrate from their ventral and dorsal VZ birth sites to peripheral regions of the cord. Furthermore although the majority of proliferating cells in the spinal cord at E16 and E18 were located in the ventral periventricular region, some proliferating cells remained in the dorsal VZ region of the cord.     

Expression pattern of LINGO-1 in the developing nervous system of the chick embryo

We isolated a chick homologue of LINGO-1 (cLINGO-1), a novel component of the Nogo-66 receptor (NgR)/p75 neurotrophin receptor (NTR) signaling complex, and examined the expression of cLINGO-1 in the developing brain and spinal cord of the chick embryo by in situ hybridization and immunohistochemistry. cLINGO-1 was expressed broadly in the spinal cord, including the ventral portion of the ventricular zone, and motor neurons. cLINGO-1 was also expressed in the dorsal root ganglion and boundary cap cells at dorsal and ventral roots. In the early embryonic brain, cLINGO-1 was first expressed in the prosencephalon and the ventral mesencephalon, and later in the telencephalon, the rostral part of the mesencephalon and some parts of the hindbrain. cLINGO-1 was also expressed in the ventral part of the neural retina and trigeminal and facial nerves. We also found that cLINGO-1, cNgR1 and p75NTR were expressed in overlapped patterns in the spinal cord and the dorsal root ganglion, but that these genes were expressed in distinct patterns in the early embryonic brain.     

Sonic hedgehog signaling is required during the appearance of spinal cord oligodendrocyte precursors

Spinal cord oligodendrocyte precursors arise in the ventral ventricular zone as a result of local signals. Ectopic oligodendrocyte precursors can be induced by sonic hedgehog (Shh) in explants of chick dorsal spinal cord over an extended developmental period. The role of Shh during normal oligodendrocyte development is, however, unclear. Here we demonstrate that Shh is localized to the ventral spinal cord immediately prior to, and during the appearance of oligodendrocyte precursors. Continued expression of Shh is required for the appearance of spinal cord oligodendrocyte precursors as neutralization of Shh signaling both in vivo and in vitro during a defined developmental period blocked their emergence. The inhibition of oligodendrocyte precursor emergence in the absence of Shh signaling was not the result of inhibiting precursor cell proliferation, and the neutralization of Shh signaling after the emergence of oligodendrocyte precursors had no effect on the appearance of additional cells or their subsequent differentiation. Similar concentrations of Shh induce motor neurons and oligodendrocytes in dorsal spinal cord explants. However, in explants from early embryos the motor neuron lineage is preferentially expanded while in explants from older embryos the oligodendrocyte lineage is preferentially expanded.     

Pathfinding during spinal tract formation in the chick-quail chimera analysed by species-specific monoclonal antibodies.

In order to analyse the spinal tract formation at early stages of development in avian embryos, chick-quail spinal cord chimeras were prepared and species-specific monoclonal antibodies (MAb) were developed. MAbs CN, QN and CQN uniquely stained chick, quail, and both chick and quail nervous tissues, respectively. All three antibodies appeared to bind to the same membrane molecule, but to different epitopes. Cord reversal revealed the features of axonal growth of both cord interneurons and dorsal root ganglion cells. Quail cord interneurons grew along an originally ventral marginal layer in the quail cord transplanted in a reversed position, then turned toward the ventral side at the boundary between the graft and the host, and grew along the host chick ventral marginal layer. Central axons of dorsal root ganglia were restricted to the ventrolateral region of the cord which originally formed the dorsal funiculus. These results suggest that cord interneurons and dorsal root ganglion cells actively select to grow along specific regions of the cord and that spinal tract formation appears to be determined by cord cells, and not by sclerotome cells.     

Olig3 Is Not Involved in the Ventral Patterning of Spinal Cord

At embryonic stages, Olig3 is initially expressed in the dorsal-most region of the spinal cord, but later in the ventral marginal zone as well. Previous studies indicated that Olig3 controlled the patterning of dorsal spinal cord and loss of Olig3 function led to the re-specification of dI2 and dI3 neurons into dI4 interneurons. However, the role of Olig3 in regulating the development of ventral spinal cord has remained unknown. BrdU labeling demonstrated that ventral Olig3 was expressed in the post-mitotic neurons and Olig3+ cells seen at late embryonic stages were born at the earlier stage but remained in the marginal zone throughout embryogenesis. Loss-of-function and gain-of-function experiment indicated that Nkx2.2 regulated the expression of Olig3 in V3 interneurons. However, Olig3 mutation didn’t apparently affect the generation and migration of ventral neurons. These findings suggest that Olig3 plays different roles in regulating the development of dorsal and ventral spinal cord.     

The location and distribution of neural crest-derived Schwann cells in developing peripheral nerves in the chick forelimb.

The location and distribution of neural crest-derived Schwann cells during development of the peripheral nerves of chick forelimbs were examined using chick-quail chimeras. Neural crest cells were labeled by transplantation of the dorsal part of the neural tube from a quail donor to a chick host at levels of the neural tube destined to give rise to brachial innervation. The ventral roots, spinal nerves, and peripheral nerves innervating the chick forelimb were examined for the presence of quail-derived neural crest cells at several stages of embryonic development. These quail cells are likely to be Schwann cells or their precursors. Quail-derived Schwann cells were present in ventral roots and spinal nerves, and were distributed along previously described neural crest migratory pathways or along the peripheral nerve fibers at all stages of development examined. During early stages of wing innervation, quail-derived Schwann cells were not evenly distributed, but were concentrated in the ventral root and at the brachial plexus. The density of neural crest-derived Schwann cells decreased distal to the plexus, and no Schwann cells were ever seen in advance of the growing nerve front. When the characteristic peripheral nerve branching pattern was first formed, Schwann cells were clustered where muscle nerves diverged from common nerve trunks. In still older embryos, neural crest-derived Schwann cells were evenly distributed along the length of the peripheral nerves from the ventral root to the distal nerve terminations within the musculature of the forelimb. These observations indicate that Schwann cells accompany axons into the developing limb, but they do not appear to lead or direct axons to their targets. The transient clustering of neural crest-derived Schwann cells in the ventral root and at places where axon trajectories diverge from one another may reflect a response to some environmental feature within these regions.     

Retention during embryonic life of the ability of avian spinal cord to induce somitic chondrogenesis in vitro

It is well established that the spinal cord of embryonic vertebrates induces sclerotomal somitic mesoderm to chondrify. We have investigated whether the spinal cord retains this inductive ability for the duration of the life of the avian embryo. Somites were isolated from embryos of H.H. stages 16 to 18 and either cultured alone in a medium which would not allow spontaneous chondrogenesis or cultured in direct contact with the spinal cord from embryos ranging in age between H.H. stages 33 and 44 (7 1/2--18 days of incubation). Somites cultured alone did not chondrify. Somites cultured in contact with either the ventral surface of the spinal cord or with the ependyma of the spinal cord chondrified in virtually 100% of all cultures--irrespective of the age of the donor embryo providing the spinal cord. The somites which were cultured in contact with the dorsal surface of the spinal cord did not undergo chondrogenesis. We conclude that the ventral spinal cord and the ependyma retain inductive ability through embryonic life and discuss the possible reasons for this.     

An analysis of dorsal root ganglia differentiation using three tissue culture systems

Summary The histogenesis of the dorsal root ganglia of chick embryos (ages 3 to 9 days) was followed in three different tissue culture systems. Organotypic explants included dorsal root ganglia connected to the lumbosacral segment of the spinal cord or isolated explants of the contralateral ganglia. Additionally, dissociated monolayer cultures of ganglia tissue were established. The gradual differentiation of progenitor neuroblasts into distinct populations of large ventrolateral and small dorsomedial neurons was observed in vivo and in vitro. Neurites developed after 3 days in the presence or absence of nerve growth factor in the medium. In contrast, autoradiographic analysis indicates that [³H]thymidine incorporation in neuronal cultures differed significantly from intact embryos. In vivo, the number of neuronal progenitor cells labeled with [³H]thymidine decreased in older embryos; in vitro, uptake of [³H]thymidine label was not observed in ganglionic progenitor cells regardless of the age of the donor embryo or the type of culture system. Lack of proliferation in ganglionic progenitor cells was not due to degeneration because vital staining and uptake of [³H]deoxyglucose indicated that neurons were metabolically active. Furthermore, the block in mitotic activity in vitro was limited to presumptive ganglionic neuronal cells. In the ependyma of the spinal cord segment connected to the dorsal root ganglia, neuronal progenitor cells were heavily labeled as were non-neuronal cells within both spinal cord and ganglia. Our results suggest that in vitro conditions can promote the differentiation of sensory neurons from early embryos (E3.5–4.5) without proliferation of progenitor cells.     

Dorsoventral differential distribution of collagen type XIV around the spinal cord is regulated by the ectoderm

Regional specification in the nervous system is a critical issue in nervous system morphogenesis. Along the dorsoventral axis of the spinal cord, ventral inductive signals of the notochord and floor plate, and dorsal ones of the epidermal ectoderm are essential. Collagen type XIV is uniquely distributed around the spinal cord with a gradient of dorsal high and ventral low at the early developmental stages of the chick embryo. In the present study it was found that collagen type XIV expression around the spinal cord was entirely regulated by the ectoderm and that even the ventralizing tissues, the notochord and floor plate, themselves could be influenced to express this molecule by the ectoderm.     

Adrenergic innervation of the extramedullary arteries of the spinal cord of cats]

The adrenergic nerves of the radical and longitudinal arteries and the dura mater at the level of cervical, thoracic and lumbar segments of the ventral and dorsal sides of the spinal cord were studied in 70 mature cats by methods of Falck and Glenner. The adrenergic fibres form developed plexuses different in the density of disposition of nerve conductors on the arteries of different segments of the spinal cord. The adrenergic fibres are also found in the pia mater tissue. Nerve fibres containing active monoaminoxidase are less in number than adrenergic ones found by Falck's method. It is probably due to activation of catecholamines being realized by other ways in addition to oxydative desamination.     

Zic1 promotes the expansion of dorsal neural progenitors in spinal cord by inhibiting neuronal differentiation

The role of Zic1 was investigated by altering its expression status in developing spinal cords. Zic genes encode zinc finger proteins homologous to Drosophila Odd-paired. In vertebrate neural development, they are generally expressed in the dorsal neural tube. Chick Zic1 was initially expressed evenly along the dorsoventral axis and its expression became increasingly restricted dorsally during the course of neurulation. The dorsal expression of Zic1 was regulated by Sonic hedgehog, BMP4, and BMP7, as revealed by their overexpressions in the spinal cord. When Zic1 was misexpressed on the ventral side of the chick spinal cord, neuronal differentiation was inhibited irrespective of the dorsoventral position. In addition, dorsoventral properties were not grossly affected as revealed by molecular markers. Concordantly, when Zic1 was overexpressed in the dorsal spinal cord in transgenic mice, we observed hypercellularity in the dorsal spinal cord. The transgene-expressing cells were increased in comparison to those of truncated mutant Zic1-bearing mice. Conversely, we observed a significant cell number reduction without loss of dorsal properties in the dorsal spinal cords of Zic1-deficient mice. Taken together, these findings suggest that Zic1 controls the expansion of neuronal precursors by inhibiting the progression of neuronal differentiation. Notch-mediated inhibition of neuronal differentiation is likely to act downstream of Zic genes since Notch1 is upregulated in Zic1-overexpressing spinal cords in both the mouse and the chick.     

Wnt/BMP signal integration regulates the balance between proliferation and differentiation of neuroepithelial cells in the dorsal spinal cord

Multiple signaling pathways regulate proliferation and differentiation of neural progenitor cells during early development of the central nervous system (CNS). In the spinal cord, dorsal signaling by bone morphogenic protein (BMP) acts primarily as a patterning signal, while canonical Wnt signaling promotes cell cycle progression in stem and progenitor cells. However, overexpression of Wnt factors or, as shown here, stabilization of the Wnt signaling component beta-catenin has a more prominent effect in the ventral than in the dorsal spinal cord, revealing local differences in signal interpretation. Intriguingly, Wnt signaling is associated with BMP signal activation in the dorsal spinal cord. This points to a spatially restricted interaction between these pathways. Indeed, BMP counteracts proliferation promoted by Wnt in spinal cord neuroepithelial cells. Conversely, Wnt antagonizes BMP-dependent neuronal differentiation. Thus, a mutually inhibitory crosstalk between Wnt and BMP signaling controls the balance between proliferation and differentiation. A model emerges in which dorsal Wnt/BMP signal integration links growth and patterning, thereby maintaining undifferentiated and slow-cycling neural progenitors that form the dorsal confines of the developing spinal cord.     

Anomalous neural differentiation induced by 5-bromo-2'-deoxyuridine during organogenesis in the rat

The influence of 5-bromo-2'-deoxyuridine (BrdU) on rat embryo development and neurogenesis was investigated using a rat conceptus culture system during organogenesis (pregnancy days 10-13). The embryos and visceral yolk sacs of conceptuses cultured with BrdU were examined for overall growth, morphological anomalies, incorporation of radiolabeled BrdU into DNA, and neurotransmitter enzyme activities in embryos. In addition, neural tubes from cultured whole embryos were isolated and mechanically dissociated into fragments and cultured again to assess neural cell differentiation into neuron-like cells. BrdU was found to incorporate differentially into embryonic and visceral yolk sac DNA with simultaneous stage-specific retardation and anomalous organogenesis in proportion to the increasing concentrations used. Neural tube differentiation of cultured embryos was markedly altered, and there were morphologically distinct neural anomalies. The neurite outgrowth from neuroblast cells (type 1) of explanted spinal neural tube fragments from BrdU-treated embryos was markedly reduced in length and number compared to those from similar areas of embryos grown without BrdU. In contrast, BrdU at the same doses did not affect differentiation of a number of neural tissue-related enzymes. These results indicate that BrdU incorporation into DNA of primordial embryonic cells significantly affects neurogenesis and differentiation of neurites from neuroblasts, which is a specific neural cytodifferentiation characteristic of neuronal cells.     

BrdU Birth Dating Can Produce Errors in Cell Fate Specification in Chick Brain Development

Birth dating neurons with bromodeoxyuridine (BrdU) labeling is an established method widely employed by neurobiologists to study cell proliferation in embryonic, postnatal, and adult brain. Birth dating studies in the chick dorsal telencephalon and the mammalian striatum have suggested that these structures develop in a strikingly similar manner, in which neurons with the same birth date aggregate to form “isochronic clusters.” Here we show that isochronic cluster formation in the chick dorsal telencephalon is an artifact. In embryos given standardly employed doses of BrdU, we observed isochronic clusters but found that clusters were absent with BrdU doses close to the limits of detection. In addition, in situ hybridization experiments established that neurons in the clusters display errors in cell type specification: BrdU cell clusters in nidopallium adopted a mesopallial neuronal fate, mesopallial clusters were misspecified as nidopallial cells, and in some instances, the BrdU clusters failed to express neuronal differentiation markers characteristic of the dorsal telencephalon. These results demonstrate that the chick dorsal telencephalon does not develop by isochronic cluster formation and highlight the need to test the integrity of BrdU-treated tissue with gene expression markers of regional and cell type identity.     

Changes in progenitor populations and ongoing neurogenesis in the regenerating chick spinal cord

The chick spinal cord can regenerate following injury until advanced developmental stages. It is conceivable that changes in stem/progenitor cell plasticity contribute to the loss of this capacity, which occurs around E13. We investigated the contribution of proliferation, phenotypic changes in radial glia progenitors, and neurogenesis to spinal cord regeneration. There was no early up-regulation of markers of gliogenic radial glia after injury either at E11 or E15. In contrast, increased proliferation in the grey matter and up-regulation of transitin expression following injury at E11, but not E15, suggested high levels of plasticity within the E11 spinal cord progenitor population that are lost by later stages. Changes in neural progenitors with development were also supported by a higher neurosphere forming ability at E11 than at E15. Co-labelling with doublecortin and neuron-specific markers and BrdU in spinal cord sections and dissociated cells showed that neurogenesis is an ongoing process in E11 chick spinal cords. This neurogenesis appeared to be complete by E15. Our findings demonstrate that the regeneration-competent chick spinal cord is less mature and more plastic than previously believed, which may contribute to its favourable response to injury, and suggest a role for neurogenesis in maintaining regenerative capacity.     

Involvement of gicerin, a cell adhesion molecule, in development and regeneration of chick sciatic nerve

We have examined the role of gicerin, an immunoglobulin superfamily cell adhesion molecule, in chick sciatic nerves during development and regeneration. Gicerin was expressed in the spinal cord, dorsal root ganglion (DRG) and sciatic nerves in embryos, but declined after hatching. Neurite extensions from explant cultures of the DRG were promoted on gicerin's ligands, which were inhibited by an anti-gicerin antibody. Furthermore, gicerin expression was upregulated in the regenerating sciatic nerves, DRG and dorsal horn of the spinal cord after injury to the sciatic nerve. These results indicate that gicerin might participate in the development and regeneration of sciatic nerves.     

The effect of nerve growth factor (NGF) on the synthesis of gangliosides

—Dorsal root ganglia from 8-day- and 14-day-old chick embryos contained gangliosides with a pattern qualitatively similar to that of embryonic chick brain. The pattern of gangliosides from dorsal root ganglia changed with age, there being a decrease in polysialogangliosides with increasing age. When isolated, dorsal root ganglia were incubated in the presence of a concentration of nerve growth factor (NGF) sufficient to promote the outgrowth of nerve fibres, there was increased incorporation of d -[1-¹⁴C]glucosamine into gangliosides. There was, however, no difference in the pattern of incorporation into gangliosides by control ganglia and those exposed to NGF.     

Perchloric acid-soluble protein regulates cell proliferation and differentiation in the spinal cord of chick embryos

The role of perchloric acid-soluble protein (PSP) was investigated in chick embryos. Fluorescently labeled anti-chick liver (CL)-PSP IgG was injected into the yolk sac in ovo at embryonic day 3, and became localized in neuroepithelial cells. Within 12 h, morphological changes were observed in 37.5% of anti-CL-PSP IgG-injected embryos, and the neuroepithelial cells formed a wavy line. No significant changes were observed in embryos injected with non-immune IgG or PBS. Increased expression of PCNA and decreased expression of neuronal class III beta-tubulin were observed in the spinal cord after anti-CL-PSP IgG injection. These results suggest that PSP controls the proliferation and differentiation of neuroepithelial cells in chick embryos.