Pleiotrophin,A Multifunctional Tumor Promoter Through Induction of Tumor Angiogenesis,Remodeling of the Tumor Microenvironment,and Activation of Stromal Fibroblasts

Pleiotrophin (PTN, Ptn) is a widely expressed, developmentally regulated 136 amino acid secreted heparin-binding cytokine. It signals through a unique signaling pathway; the PTN receptor is the transmembrane receptor protein tyrosine phosphatase (RPTP)β/ζ. RPTPβ/ζ is inactivated by PTN, which leads to increased tyrosine phosphorylation of the downstream targets of the PTN/RPTPβ/ζ signaling pathway. Pleiotrophin gene expression is found in cells in early differentiation during different developmental periods. It is upregulated in cells with an early differentiation phenotype in wound repair. The Ptn gene also is a proto-oncogene; PTN is expressed in human tumor cells, and, in cell lines derived from human tumors that express Ptn, Ptn expression is constitutive and thus "inappropriate". Importantly, properties of different cells induced by PTN in PTN-stimulated cells are strikingly similar to properties of highly malignant cells. Furthermore, transformed cells into which Ptn is introduced undergo "switches" to malignant cells of higher malignancy with properties that are strikingly similar to properties of PTN-stimulated cells. These unique features of PTN support the conclusion that constitutive PTN signaling in malignant cells that inappropriately express Ptn functions as a potent tumor promoter. Recently, in confirmation, Ptn targeted by the mouse mammary tumor virus (MMTV) promoter in a transgenic mouse model was found to promote breast cancers to a more aggressive breast cancer cell phenotype that morphologically closely resembles scirrhous carcinoma in human; in addition, it promoted a striking increase in tumor angiogenesis and a remarkable degree of remodeling of the micro-environment. Pleiotrophin thus regulates both different normal and pathological functions; collectively, the different studies have uncovered the unique ability of a single cytokine PTN, which signals through the unique PTN/RPTPβ/ζ signaling pathway, to induce the many properties associated with tumor promotion in the malignant cells that constitutively express Ptn and in their microenvironment.     

Pleiotrophin is expressed in avian somites and tendon anlagen

Pleiotrophin (Ptn) is a secreted, developmentally regulated growth factor associated with the extracellular matrix. During mammalian embryogenesis, Ptn has been suggested to play a role in the development of various embryonic structures including nervous system and skeleton. In the avian embryo, Ptn has been proposed to be involved in limb cartilage development, but embryonic Ptn expression has not been comprehensively studied. We isolated a cDNA fragment containing the full-length coding sequence of chick Ptn and studied the expression of Ptn in detail until embryonic day 10. We, furthermore, isolated a 6,385-bp phage clone containing the Ptn cDNA of 2,551 bp and additional 3,787 bp downstream of the published Ptn cDNA sequence classifying a yet Ptn-unrelated chEST clone as the 3′ untranslated region of Ptn. Our studies revealed novel expression domains in developing somites and during limb formation. We found prominent expression in the somitocoel cells of epithelial somites, and in a sclerotomal subcompartment, the syndetome, which gives rise to the axial tendons in the vertebral motion segment. In the limbs, Ptn was markedly expressed in tendon anlagen and in phalangeal joints. Our results introduce Ptn as a novel marker gene in avian somite and tendon development.     

Regulation of SC1/DM-GRASP during the migration of motor neurons in the chick embryo brain stem

The hindbrain of the chick embryo contains three classes of motor neurons: somatic, visceral, and branchial motor. During development, somata of neurons in the last two classes undergo a laterally directed migration within the neuroepithelium; somata translocate towards the nerve exit points, through which motor axons are beginning to extend into the periphery. All classes of motor neuron are immunopositive for the SC1/DM-GRASP cell surface glycoprotein. We have examined the relationship between patterns of motor neuron migration, axon outgrowth, and expression of the SC1/DM-GRASP mRNA and protein, using anterograde or retrograde axonal tracing, immunohistochemistry, and in situ hybridization. We find that as motor neurons migrate laterally, SC1/DM-GRASP is down-regulated, both on neuronal somata and axonal surfaces. Within individual motor nuclei, these lateral, more mature neurons are found to possess longer axons than the young, medial cells of the population. Labelling of sensory or motor axons growing into the second branchial arch also shows that motor axons reach the muscle plate first, and that SC1/DM-GRASP is expressed on the muscle at the time growth cones arrive. 1994 John Wiley & Sons, Inc.     

Ultrastructure of the circumoral nerve ring and the radial nerve cords in holothurians (Echinodermata)

The circumoral nerve ring and the radial nerve cords (RNCs) of Eupentacta fraudatrix and Pseudocnus lubricus (Holothuroidea) were examined as an example of holothurian nervous tissue. The RNC is composed of outer ectoneural and inner hyponeural layers, which are interconnected with one another via short neural bridges. The circumoral nerve ring is purely ectoneural. Both ectoneural and hyponeural components are epithelial tubes with a thick neuroepithelium at one side. A thin ciliated non-neuronal epithelium complements the neuroepithelium to form a tube, thereby enclosing the epineural and hyponeural canals. The whole of the ectoneural and hyponeural subsystems is separated from the surrounding tissue by a continuous basal lamina. The nerve ring and the ectoneural and hyponeural parts of the radial nerves are all neuroepithelia composed of supporting cells and neurons. Supporting cells are interpreted as being glial cells. Based on ultrastructural characters, three types of neurons can be distinguished: (1) putative primary sensory neurons, whose cilium protrudes into the epineural or hyponeural canal; (2) non-ciliated neurons with swollen rough endoplasmic reticulum cisternae; (3) monociliated neurons that are embedded in the trunk of nerve fibers. Different types of synapses occur in the neuropile area. They meet all morphological criteria of classical chemical synapses. Vacuolated cells occur in the neuroepithelium of E. fraudatrix, but are absent in P. lubricus; their function is unknown. The cells of the non-neuronal epithelia that overlie the ectoneural and hyponeural canals are hypothesized to belong to the same cell type as the supporting cells of the neuroepithelium.     

slit: an EGF-homologous locus of D. melanogaster involved in the development of the embryonic central nervous system

A family of loci homologous to the EGF-like portion of Notch, a gene involved in neurogenesis, have been identified in D. melanogaster. The sequence, spatial, and temporal distribution of both RNA and protein of one of these loci suggest a possible role in the development of the central nervous system (CNS). In situ hybridization and antibody staining of embryos show initial localization in cells along the midline of the neuroepithelium. High level expression is restricted in the developing embryo to a subset of six midline glial cells abutting growing axons. Extracellular localization is suggested by the presence of EGF-like repeats in the deduced protein sequence and antibody staining. Cytological, immunocytochemical, genetic, and molecular data show that this gene corresponds to the slit locus. Mutations in this locus result in the collapse of the regular scaffold of commissural and longitudinal axon tracts in the embryonic central nervous system.     

Embryonic brain tract formation in Drosophila melanogaster

 During embryogenesis in insects, the axonscaffold of the brain is built around the embryonic foregut which separates the anlagen of the brain hemispheres. Here, we investigate this process in Drosophila and show that the major longitudinal and horizontal tracts of the embryonic brain form superficially near the interface between the foregut and embryonic brain hemispheres. We identify three types of cellular structures which might be involved in tract formation. These are rows of glial cells at the medial brain margin, cellular bridges composed of neuronal somata and the epithelial surface of the foregut itself. The close proximity to the outgrowing axons suggests that the structures at the brain-foregut interface may play a role in the morphogenesis of embryonic brain tracts in Drosophila. Received: 11 November 1996 / Accepted: 3 January 1997     

Miple1 and miple2 encode a family of MK/PTN homologues in Drosophila melanogaster

Midkine (MK) and Pleiotrophin (PTN) are small heparin-binding cytokines with closely related structures. To date, this family of proteins has been implicated in multiple processes, such as growth, survival, and migration of various cells, and has roles in neurogenesis and epithelial–mesenchymal interaction during organogenesis. In this report, we have characterized two members of the MK/PTN family of proteins in Drosophila, named Miple1 and Miple2, from Midkine and Pleiotrophin. Drosophila miple1 and miple2 encode secreted proteins which are expressed in spatially restricted, nonoverlapping patterns during embryogenesis. Expression of miple1 can be found at high levels in the central nervous system, while miple2 is strongly expressed in the developing midgut endoderm. The identification of homologues of the MK/PTN family in this genetically tractable model organism should allow an analysis of their function during complex developmental processes. C. Englund, A. Birve, and L. Falileeva contributed equally to this work.     

Base composition, selection, and phylogenetic significance of indels in the recombination activating gene-1 in vertebrates

Background

Echinoderms and chordates belong to the same monophyletic taxon, the Deuterostomia. In spite of significant differences in body plan organization, the two phyla may share more common traits than was thought previously. Of particular interest are the common features in the organization of the central nervous system. The present study employs two polyclonal antisera raised against bovine Reissner's substance (RS), a secretory product produced by glial cells of the subcomissural organ, to study RS-like immunoreactivity in the central nervous system of sea cucumbers.

Results

In the ectoneural division of the nervous system, both antisera recognize the content of secretory vacuoles in the apical cytoplasm of the radial glia-like cells of the neuroepithelium and in the flattened glial cells of the non-neural epineural roof epithelium. The secreted immunopositive material seems to form a thin layer covering the cell apices. There is no accumulation of the immunoreactive material on the apical surface of the hyponeural neuroepithelium or the hyponeural roof epithelium. Besides labelling the supporting cells and flattened glial cells of the epineural roof epithelium, both anti-RS antisera reveal a previously unknown putative glial cell type within the neural parenchyma of the holothurian nervous system.

Conclusion

Our results show that: a) the glial cells of the holothurian tubular nervous system produce a material similar to Reissner's substance known to be synthesized by secretory glial cells in all chordates studied so far; b) the nervous system of sea cucumbers shows a previously unrealized complexity of glial organization. Our findings also provide significant clues for interpretation of the evolution of the nervous system in the Deuterostomia. It is suggested that echinoderms and chordates might have inherited the RS-producing radial glial cell type from the central nervous system of their common ancestor, i.e., the last common ancestor of all the Deuterostomia.     

Conserved and novel functions for Netrin in the formation of the axonal scaffold and glial sheath cells in spiders

Netrins are well known for their function as long-range chemotropic guidance cues, in particular in the ventral midline of vertebrates and invertebrates. Over the past years, publications are accumulating that support an additional short-range function for Netrins in diverse developmental processes such as axonal pathfinding and cell adhesion. We describe here the formation of the axonal scaffold in the spiders Cupiennius salei and Achaearanea tepidariorum and show that axonal tract formation seems to follow the same sequence as in insects and crustaceans in both species. First, segmental neuropiles are established which then become connected by the longitudinal fascicles. Interestingly, the commissures are established at the same time as the longitudinal tracts despite the large gap between the corresponding hemi-neuromeres which results from the lateral movement of the germband halves during spider embryogenesis. We show that Netrin has a conserved function in the ventral midline in commissural axon guidance. This function is retained by an adaptation of the expression pattern to the specific morphology of the spider embryo. Furthermore, we demonstrate a novel function of netrin in the formation of glial sheath cells that has an impact on neural precursor differentiation. Loss of Netrin function leads to the absence of glial sheath cells which in turn results in premature segregation of neural precursors and overexpression of the early motor- and interneuronal marker islet. We suggest that Netrin is required in the differentiated sheath cells for establishing and maintaining the interaction between NPGs and sheath cells. This short-range adhesive interaction ensures that the neural precursors maintain their epithelial character and remain attached to the NPGs. Both the conserved and novel functions of Netrin seem to be required for the proper formation of the axonal scaffold.     

Developmental distribution of glycosaminoglycans in embryonic rat brain: Relationship to axonal tract formation

Glycosaminoglycans, the sugar moieties of proteoglycans, modulate axonal growth in vitro. However, their anatomical distribution in relation to developing axonal tracts in the rat brain has not been studied. Here, we examined the immunohistochemical distribution of chondroitin-6-sulfate and chondroitin-4-sulfate, two related glycosaminoglycan epitopes, which are present in three types of glycosaminoglycans: chondroitin sulfate C, chondroitin sulfate A, and chondroitin sulfate B. Further, we compared their distribution pattern to that of axonal tract development. Both glycosaminoglycan epitopes showed a heterogeneous spatiotemporal distribution within the developing rat brain. However, the expression of chondroitin-4-sulfate was more restricted than that of chondroitin-6-sulfate, although both epitopes were detected from embryonic day 13 until the day of birth, overlapping in many regions of the central nervous system including cortex, hippocampus, thalamus, and hindbrain. After birth, the levels of expression of both glycosaminoglycan epitopes progressively decreased and were practically undetectable after the first postnatal week. The expression of chondroitin-6-sulfate and, to a lesser extent, that of chondroitin-4-sulfate, was preferentially associated to the extracellular matrix surrounding specific axon bundles. However, the converse association was not true, and several apparently similar types of axon developed on a substrate devoid of both types of glycosaminoglycan epitopes. These results provide an anatomical background for the idea that different types of glycosaminoglycans may contribute to establish the complex set of guidance cues necessary for the specific development of defined axon tracts in the central nervous system. © 1996 John Wiley & Sons, Inc.     

Glial patterning during postembryonic development of central neuropiles in the brain of the honeybee

 Glial cells are involved in several functions during the development of the nervous system. To understand potential glial contributions to neuropile formation, we examined the cellular pattern of glia during the development of the mushroom body, antennal lobe and central complex in the brain of the honeybee. Using an antibody against the glial-specific repo-protein of Drosophila, the location of the glial somata was detected in the larval and pupal brain of the bee. In the early larva, a continuous layer of glial cell bodies defines the boundaries of all growing neuropiles. Initially, the neuropiles develop in the absence of any intrinsic glial somata. In a secondary process, glial cells migrate into defined locations in the neuropiles. The corresponding increase in the number of neuropile-associated glial cells is most likely due to massive immigrations of glial cells from the cell body rind using neuronal fibres as guidance cues. The combined data from the three brain regions suggest that glial cells can prepattern the neuropilar boundaries. Received: 3 November 1996 / Accepted: 7 February 1997     

WALLERIAN DEGENERATION IN RABBIT OPTIC NERVE: CELLULAR LOCALIZATION IN THE CENTRAL NERVOUS SYSTEM OF THE S-100 AND 14-3-2 PROTEINS1

Abstract— The S-100 and 14-3-2 proteins, which are found only in nervous tissues, were measured in degenerating rabbit optic nerve at 0, 5 10, 20, 40, 60, 80, 100, 150 and 200 days after unilateral enucleation in order to obtain indications of the cellular localization of these proteins in the central nervous system. S-100 increased and 14-3-2 decreased (both approximately 70 per cent) in cut nerves by 200 days of degeneration. Changes in amounts of the proteins were related to cellular alterations which characterize the degenerative process, as demonstrated by electron microscopy. In uncut nerves (intact eye) from these experimental animals, S-100 increased and 14-3-2 decreased slightly at 5 days, after which time the levels of each returned to those approximating the content in corresponding nerves from unoperated control animals. No appreciable change in total soluble proteins was measured in degenerating or intact nerves. Since S-100 increased and 14-3-2 decreased in the degenerating optic nerve as it became relatively enriched in glial constituents but impoverished in axonal content, it is suggested that S-100 is primarily a glial protein and 14-3-2 predominantly a neuronal protein in the central nervous system.     

An ultrastructural study of peripheral neurons and associated non-neural structures in the bivalve mollusc, Spisula solidissima

The ultrastructural morphology of peripheral neurons and associated structures in the bivalve mollusc, Spisula solidissima have been studied in an effort to describe the synaptic topography and to provide anatomical correlates of previous physiological observations. The somata of the peripheral neurons are located within the perineurium at branch points of the siphonal nerves. There are many fiber-fiber synaptic contacts which are characterized by isolated sites of contact with membrane specialization and unilateral accumulation of synaptic vesicles. There are also synaptic contacts involving the somata, both axo-somatic and somato-axonic, the two being distinguishable on the basis of the polarity of vesicle accumulation. All of the observed synaptic profiles were similar in appearance regardless of the neuron loci involved. Much of the non-synaptic soma surface is covered with processes of glial cells. Likewise, in many cases, individual fibers and groups of fibers are encased with glial processes. Unique clusters of membrane bound, pigment containing glial like cells occur throughout the nervous system of Spisula. The heterogeneous appearance of the inclusions and the presence of lysosome-like bodies in the cytoplasm of these cells suggest a possible phagocytic role.     

Polydatin promotes the neuronal differentiation of bone marrow mesenchymal stem cells in vitro and in vivo: Involvement of Nrf2 signalling pathway

Bone marrow mesenchymal stem cell (BMSC) transplantation represents a promising repair strategy following spinal cord injury (SCI), although the therapeutic effects are minimal due to their limited neural differentiation potential. Polydatin (PD), a key component of the Chinese herb Polygonum cuspidatum, exerts significant neuroprotective effects in various central nervous system disorders and protects BMSCs against oxidative injury. However, the effect of PD on the neuronal differentiation of BMSCs, and the underlying mechanisms remain inadequately understood. In this study, we induced neuronal differentiation of BMSCs in the presence of PD, and analysed the Nrf2 signalling and neuronal differentiation markers using routine molecular assays. We also established an in vivo model of SCI and assessed the locomotor function of the mice through hindlimb movements and electrophysiological measurements. Finally, tissue regeneration was evaluated by H&E staining, Nissl staining and transmission electron microscopy. PD (30 μmol/L) markedly facilitated BMSC differentiation into neuron‐like cells by activating the Nrf2 pathway and increased the expression of neuronal markers in the transplanted BMSCs at the injured spinal cord sites. Furthermore, compared with either monotherapy, the combination of PD and BMSC transplantation promoted axonal rehabilitation, attenuated glial scar formation and promoted axonal generation across the glial scar, thereby enhancing recovery of hindlimb locomotor function. Taken together, PD augments the neuronal differentiation of BMSCs via Nrf2 activation and improves functional recovery, indicating a promising new therapeutic approach against SCI.     

VEGF in the nervous system

Vascular endothelial growth factor (VEGF, VEGFA) is critical for blood vessel growth in the developing and adult nervous system of vertebrates. Several recent studies demonstrate that VEGF also promotes neurogenesis, neuronal patterning, neuroprotection and glial growth. For example, VEGF treatment of cultured neurons enhances survival and neurite growth independently of blood vessels. Moreover, evidence is emerging that VEGF guides neuronal migration in the embryonic brain and supports axonal and arterial co-patterning in the developing skin. Even though further work is needed to understand the various roles of VEGF in the nervous system and to distinguish direct neuronal effects from indirect, vessel-mediated effects, VEGF can be considered a promising tool to promote neuronal health and nerve repair.

Note: Previously published in VEGF in Development, edited by Christiana Ruhrberg. Landes Bioscience and Springer Science+Business Media 2008; pp. 91-103.     

Dual roles of the chemorepellent axon guidance molecule RGMa in establishing pioneering axon tracts and neural fate decisions in embryonic vertebrate forebrain

Repulsive guidance molecule A (RGMa) is a glycosylphosphatidylinositol‐anchored plasma membrane protein that was originally identified based on its chemorepulsive activity during axon navigation in the developing nervous system. Knock down of RGMa has previously shown to perturb axon navigation in the developing Xenopus forebrain (Wilson and Key, 2006). In order to further understand the in vivo role of RGMa in axon guidance, we have adopted an in vivo gain‐of‐function approach. RGMa was mosaically overexpressed in the developing Xenopus embryo by the injection of mRNA into single blastomeres. Ectopic expression of RGMa affected the morphology and the topography of developing axon tracts in vivo. Pioneer axons misrouted or aberrantly projected in response to ectopic RGMa in the developing Xenopus forebrain, confirming the in vivo chemorepulsive activity of this ligand. In addition, we show here for the first time that overexpression of RGMa acts cell‐autonomously to generate ectopic neurons in the developing embryonic brain. Taken together, the current study reveals a pleiotropic role of RGMa in early vertebrate embryonic brain in the spatial organization of axon tracts, pioneer axon guidance, and neural cell differentiation. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

Valproic acid stimulates proliferation of glial precursors during cortical gliogenesis in developing rat

Valproic acid (VPA) is a neurotherapeutic drug prescribed for seizures, bipolar disorder, and migraine, including women of reproductive age. VPA is a well‐known teratogen that produces congenital malformations in many organs including the nervous system, as well as later neurodevelopmental disorders, including mental retardation and autism. In developing brain, few studies have examined VPA effects on glial cells, particularly astrocytes. To investigate effects on primary glial precursors, we developed new cell culture and in vivo models using frontal cerebral cortex of postnatal day (P2) rat. In vitro, VPA exposure elicited dose‐dependent, biphasic effects on DNA synthesis and proliferation. In vivo VPA (300 mg/kg) exposure from P2 to P4 increased both DNA synthesis and cell proliferation, affecting primarily astrocyte precursors, as >75% of mitotic cells expressed brain lipid‐binding protein. Significantly, the consequence of early VPA exposure was increased astrocytes, as both S100‐β+ cells and glial fibrillary acidic protein were increased in adolescent brain. Molecularly, VPA served as an HDAC inhibitor in vitro and in vivo as enhanced proliferation was accompanied by increased histone acetylation, whereas it elicited changes in culture in cell‐cycle regulators, including cyclin D1 and E, and cyclin‐dependent kinase (CDK) inhibitors, p21 and p27. Collectively, these data suggest clinically relevant VPA exposures stimulate glial precursor proliferation, though at higher doses can elicit inhibition through differential regulation of CDK inhibitors. Because changes in glial cell functions are proposed as mechanisms contributing to neuropsychiatric disorders, these observations suggest that VPA teratogenic actions may be mediated through changes in astrocyte generation during development. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 780–798, 2016     

Migration patterns of sympathetic preganglionic neurons in embryonic rat spinal cord

The displacement of immature neurons from their place of origin in the germinal epithelium toward their adult positions in the nervous system appears to involve migratory pathways or guides. While the importance of radial glial fibers in this process has long been recognized, data from recent investigations have suggested that other mechanisms might also play a role in directing the movement of young neurons. We have labeled autonomic preganglionic cells by microinjections of horseradish peroxidase (HRP) into the sympathetic chain ganglia of embryonic rats in order to study the migration and differentiation of these spinal cord neurons. Our results, in conjunction with previous observations, suggest that the migration pattern of preganglionic neurons can be divided into three distinct phases. In the first phase, the autonomic motor neurons arise in the ventral ventricular zone and migrate radially into the ventral horn of the developing spinal cord, where, together with somatic motor neurons, they form a single, primitive motor column (Phelps P. E., Barber R. P., and Vaughn J. E. (1991). J. Comp. Neurol. 307:77–86). During the second phase, the autonomic motor neurons separate from the somatic motor neurons and are displaced dorsally toward the intermediate spinal cord. When the preganglionic neurons reach the intermediolateral (IML) region, they become progressively more multipolar, and many of them undergo a change in alignment, from a dorsoventral to a mediolateral orientation. In the third phase of autonomic motor neuron development, some of these cells are displaced medially, and occupy sites between the IML and central canal. The primary and tertiary movements of the preganglionic neurons are in alignment with radial glial processes in the embryonic spinal cord, an arrangement that is consistent with a hypothesis that glial elements might guide autonomic motor neurons during these periods of development. In contrast, during the second phase, the dorsal translocation of preganglionic neurons occurs in an orientation perpendicular to radial glial fibers, indicating that glial elements are not involved in the secondary migration of these cells. The results of previous investigations have provided evidence that, in addition to glial processes, axonal pathways might provide a substrate for neuronal migration. Logically, therefore, it is possible that the secondary dorsolateral translocation of autonomic preganglionic neurons could be directed along early forming circumferential axons of spinal association interneurons, and this hypothesis is supported by the fact that such fibers are appropriately arrayed in both developmental time and space to guide this movement.