Since tenascin may influence neuronal cell development, we studied its expression pattern using immunocytochemistry, in situ hybridization, Northern blot analysis, and immunochemistry in the developing and adult mouse cerebellar cortex. Tenascin immunoreactivity was detectable in all layers of the developing cerebellar cortex. In the external granular layer, only the radially oriented processes of Golgi epithelial cells were immunoreactive, whereas the densely packed cell bodies were immunonegative. Tenascin was hardly detectable at contact sites between migrating granule cells and processes of Golgi epithelial cells. Axons of granule cells in the molecular layer were immunoreactive, whereas their cell bodies in the internal granular layer lacked detectable levels of tenascin. By in situ hybridization, only Golgi epithelial cells and astrocytes of the internal granular layer and prospective white matter, but not nerve cells, could be shown to synthesize detectable levels of tenascin mRNA in the developing mouse cerebellar cortex. Thus, tenascin in the cerebellar cortex seems to be a glia-derived molecule that becomes adsorbed to neuronal surfaces in a topographically restricted pattern in situ. Levels of tenascin protein and mRNA decreased significantly with increasing age. In the adult, tenascin immunoreactivity was weak and mainly restricted to the molecular layer and tenascin mRNA was confined to Golgi epithelial cells, indicative for a functional heterogeneity in differentiated cerebellar astrocytes. Quantitative immunoblot analysis revealed that the 225 and 240 kDa components of tenascin were developmentally downregulated at a faster rate than the 190 and 200 kDa components, corresponding to the faster downregulation of the 8 kilobase (kb) mRNA species compared to the 6 kb mRNA species as revealed by Northern blot analysis. These observations indicate a differentially regulated expression of the tenascin components. We hypothesize that glia-derived tenascin modifies the functional properties of nerve cell surfaces and that tenascin is involved in such different morphogenetic events as neurite growth and oligodendrocyte distribution.

The MAG-deficient mouse was used to test whether MAG acts as a significant inhibitor of axonal regeneration in the adult mammalian CNS, as suggested by cell culture experiments. Cell spreading, neurite elongation, or growth cone collapse of different cell types in vitro was not significantly different when myelin preparations or optic nerve cryosections from either MAG-deficient or wild-type mice were used as a substrate. More importantly, the extent of axonal regrowth in lesioned optic nerve and corticospinal tract in vivo was similarly poor in MAG-deficient and wild-type mice. However, axonal regrowth increased significantly and to a similar extent in both genotypes after application of the IN-1 antibody directed against the neurite growth inhibitors NI-35 and NI-250. These observations do not support the view that MAG is a significant inhibitor of axonal regeneration in the adult CNS.

The molecular determinants controlling the topographically restricted distribution of neural cells in the mammalian CNS are largely unknown. In the mouse, myelin-forming oligodendrocytes are differentially distributed along retinal ganglion cell axons. These axons are myelin free intraretinally and in the most proximal (i.e., retinal) part of the optic nerve, but become myelinated in the distal (i.e., chiasmal) part of the optic nerve. Tenascin protein and mRNA are detectable in increased amounts at the retinal end of the developing optic nerve before the arrival of oligodendrocyte progenitor cells and are restricted to this region in the adult optic nerve. Tenascin is a nonadhesive substrate for oligodendrocytes and their progenitor cells in vitro when offered as a substrate in choice with polyornithine. These observations suggest that tenascin is critical for the establishment and maintenance of the restricted distribution of myelin-forming oligodendrocytes along retinal ganglion cell axons of the mouse.

The analysis of mice deficient in the myelin-associated glycoprotein (MAG) or Fyn, a nonreceptor-type tyrosine kinase proposed to act as a signaling molecule downstream of MAG, has revealed that both molecules are involved in the initiation of myelination. To obtain more insights into the role of the MAG-Fyn signaling pathway during initiation of myelination and formation of morphologically intact myelin sheaths, we have analyzed optic nerves of MAG-, Fyn-and MAG/Fyn-deficient mice. We observed a slight hypomyelination in optic nerves of MAG mutants that was significantly increased in Fyn mutants and massive in MAG/Fyn double mutants. The severe morphological phenotype of MAG/Fyn mutants, accompanied by behavioral deficits, substantiates the importance of both molecules for the initiation of myelination. The different severity of the phenotype of different genotypes indicates that the MAG-Fyn signaling pathway is complex and suggests the presence of compensatory mechanisms in the single mutants. However, data are also compatible with the possibility that MAG and Fyn act independently to initiate myelination. Hypomyelination of optic nerves was not related to a loss of oligodendrocytes, indicating that the phenotype results from impaired interactions between oligodendrocyte processes and axons and/or impaired morphological maturation of oligodendrocytes. Finally, we demonstrate that Fyn, unlike MAG, is not involved in the formation of ultrastructurally intact myelin sheaths. Key words: double knock-out mutant; hypomyelination; Fyn; MAG; oligodendrocyte; optic nerve; spinal cordMyelin sheaths electrically insulate axons, and the generation of action potentials is thus confined to myelin-free regions of axons, the nodes of Ranvier. The resulting saltatory conduction of nerve impulses increases the speed at which information is propagated along axons. Given the functional importance of myelin, it is of particular interest to understand the differentiation of myelinating glial cells, the interaction between myelin-forming glial cells and axons, and the formation and maintenance of myelin at the molecular level.One of the molecules involved in the formation and maintenance of myelin is the myelin-associated glycoprotein (MAG), a member of the immunoglobulin superfamily (Arquint et al., 1987;Lai et al., 1987;Salzer et al., 1987). The analysis of MAG-deficient mice has revealed abnormal interactions between myelin-forming glial cells and axons and a variety of ultrastructural abnormalities of myelin sheaths in the CNS, and degeneration of myelin and axons in the peripheral nervous system (PNS) (Li et al., 1994;Montag et al., 1994) (for review, see Schachner and Bartsch, 2000). Of particular interest for the present study is a delayed myelination of developing optic nerves of MAG-deficient mice (Montag et al., 1994) and a hypomyelination of optic nerves of adult null mutants . Myelination in the PNS, in contrast, was not retarded in the absence of MAG (Montag et al., 1994). All these results demonstrate that MAG performs...

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Key Words: HOXC9 Ⅲ interleukin 8 Ⅲ vascular morphogenesis Ⅲ zebrafish T he vascular system is the first organ during embryonic development and is mainly formed by 2 processes called vasculogenesis and angiogenesis. 1 A number of important signaling molecules and pathways have been identified in the past 2 decades, such as the vascular endothelial growth factors (VEGFs), ephrins, angiopoietins, netrins, chemokines, and their receptors, regulating different processes of vasculogenesis and angiogenesis. 1 In addition, several upstream transcriptional regulators of these pathways have been identified and functionally characterized. Examples for endothelial-acting transcription factors are Tal1, Gata2, Forkhead, Krüppel-like factor, Ets proteins, Hey genes, Coup-TFII, and Prox1 mediating vascular processes of hematopoietic and endothelial transcription, differentiation, maturation, remodeling, specification, arteriovenous differentiation, and lymphatic patterning. 2 Homeobox genes are characterized by a 61-amino-acidlong homeodomain. These are transcription factors regulating important developmental processes. Beyond their original identification as essential regulators of spatial body development in Drosophila and in other organisms, HOX genes have more recently been recognized as transcription factors regulating physiological and pathophysiological processes in the adult. 3 HOX genes are organized in mice and humans in 4 clusters consisting of 39 different genes. According to sequence homology and genomic position, 13 paralogous groups have been defined. HOX genes regulate gene expression in various developmental processes in higher vertebrates according to 3 basic principles, named spatial collinearity, temporal collinearity, and posterior prevalence. 3 Potential roles of different HOX genes in vascular development and function are poorly studied, and molecular targets of HOX genes in the vasculature are mostly unknown. Yet a number of seminal reports have suggested that different HOX genes regulate various vascular processes, such as vasculogenesis, angiogenesis, vessel maturation, endothelial prolifOriginal received March 25, 2011; revision received April 5, 2011; accepted April 5, 2011. In March 2011 Using a combination of several cellular and in vivo experiments, the experiments of this study have identified HOXC9 as a major negative regulator of angiogenesis in vitro and of vascular development in zebrafish by repressing IL-8 functions. In HUVEC, HOXC9 is highly upregulated in resting endothelial cells. It is downregulated during hypoxic episodes, and HOXC9 represses angiogenic activation of endothelial cells. In zebrafish, HOXC9 overexpression inhibits vascular development, and expression silencing of its target gene IL-8 recapitulates a similar vascular phenotype. Therefore, the data identify HOXC9 as an endothelial cell active transcription factor promoting the resting, antiangiogenic endothelial cell phenotype in an IL-8 -dependent manner. Non-standard Abbreviations and Acronyms MethodsEmbryos of ...

We recently reported that some retinal ganglion cell axons in mice deficient for the myelin-associated glycoprotein are concentrically surrounded by more than one myelin sheath. In the present study, we demonstrate that myelin sheaths displaced from the axon reveal a normal ultrastructure of compact myelin, with the only exception that multiple myelination of axons frequently correlates with the presence of unfused regions of major dense lines. Supernumerary sheaths terminated on other sheaths or on astrocyte cell surfaces in a pattern closely resembling the morphology of a true paranode. The thickness of compact myelin of multiply myelinated axons was significantly increased when compared with axons of similar caliber surrounded by a single myelin sheath. Our observations demonstrate that maintenance of compact myelin and paranodal regions is not dependent on direct axonal contact and that the presence of more than one concentric myelin sheath around an axon results in dysregulation of the axon-to-fiber ratio.

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