A mesenchymal-like ZEB1(+) niche harbors dorsal radial glial fibrillary acidic protein-positive stem cells in the spinal cord.

In humans and rodents the adult spinal cord harbors neural stem cells located around the central canal. Their identity, precise location, and specific signaling are still ill-defined and controversial. We report here on a detailed analysis of this niche. Using microdissection and glial fibrillary acidic protein (GFAP)-green fluorescent protein (GFP) transgenic mice, we demonstrate that neural stem cells are mostly dorsally located GFAP(+) cells lying ependymally and subependymally that extend radial processes toward the pial surface. The niche also harbors doublecortin protein (Dcx)(+) Nkx6.1(+) neurons sending processes into the lumen. Cervical and lumbar spinal cord neural stem cells maintain expression of specific rostro-caudal Hox gene combinations and the niche shows high levels of signaling proteins (CD15, Jagged1, Hes1, differential screening-selected gene aberrative in neuroblastoma [DAN]). More surprisingly, the niche displays mesenchymal traits such as expression of epithelial-mesenchymal-transition zinc finger E-box-binding protein 1 (ZEB1) transcription factor and smooth muscle actin. We found ZEB1 to be essential for neural stem cell survival in vitro. Proliferation within the niche progressively ceases around 13 weeks when the spinal cord reaches its final size, suggesting an active role in postnatal development. In addition to hippocampus and subventricular zone niches, adult spinal cord constitutes a third central nervous system stem cell niche with specific signaling, cellular, and structural characteristics that could possibly be manipulated to alleviate spinal cord traumatic and degenerative diseases.

Neurosphere-derived neural cells show region-specific behaviour in vitro.

Neurosphere cultures provide a useful model to study neural stem/progenitor cells (NSC/NPCs). The degree to which neurospheres (NS) retain their regional identity in vitro has, however, been questioned. Here, NS obtained from mouse embryonic cortex, striatum or spinal cord were compared after differentiation. Neurons from cortical NS formed well ordered clusters containing astrocytes, those from striatal NS formed an external ring at the borderof the astrocyte layer, whereas those from spinal cord NS spread radially like the astrocytes. Such in-vitro neural behaviour was region-specific and persisted in clonal conditions, providing evidence of the maintenance of positional cues in NS cultures.

Macrophage contribution to the response of the rat organ of Corti to amikacin.

Transdifferentiation of nonsensory supporting cells into sensory hair cells occurs naturally in the damaged avian inner ear. Such transdifferentiation was achieved experimentally in the cochlea of deaf guinea pigs through Atoh 1 gene transfection. Supporting cells may therefore serve as targets for transdifferentiation therapy. Supporting cells rapidly degenerate after hair cell disappearance, however, limiting the therapeutic window for gene transfer. We studied the time course of ultrastructural and phenotypical changes occurring in Deiters cells (hair cell supporting cells) after ototoxic treatment in the rat. The presence of macrophages in the cochlea was also investigated, to study any deleterious effects they may have on pathologic tissues. One week after treatment most hair cells had disappeared. Deiters cells no longer expressed the glial marker vimentin but instead displayed typical hair cell markers, the calcium binding proteins calbindin and parvalbumin. This suggests that a process of transdifferentiation of Deiters cells into hair cells was activated. By 3 weeks post-treatment, however, the Deiters cells began to degenerate and by 10 weeks post-treatment the organ of Corti was degraded fully. Interestingly, a marked increase in macrophage density was seen after the end of amikacin treatment to 10 weeks post-treatment. This suggests chronic inflammation is involved in epithelium degeneration. Consequently, early treatments with anti-inflammatory factors might promote supporting cell survival, thus improving the efficacy of more specific strategies aimed to regenerate hair cells from nonsensory cells.

NG2 and Olig2 expression provides evidence for phenotypic deregulation of cultured central nervous system and peripheral nervous system neural precursor cells.

Neural stem cells cultured with fibroblast growth factor 2 (FGF2)/epidermal growth factor (EGF) generate clonal expansions called neurospheres (NS), which are widely used for therapy in animal models. However, their cellular composition is still poorly defined. Here, we report that NS derived from several embryonic and adult central nervous system (CNS) regions are composed mainly of remarkable cells coexpressing radial glia markers (BLBP, RC2, GLAST), oligodendrogenic/neurogenic factors (Mash1, Olig2, Nkx2.2), and markers that in vivo are typical of the oligodendrocyte lineage (NG2, A2B5, PDGFR-alpha). On NS differentiation, the latter remain mostly expressed in neurons, together with Olig2 and Mash1. Using cytometry, we show that in growing NS the small population of multipotential self-renewing NS-forming cells are A2B5(+) and NG2(+). Additionally, we demonstrate that these NS-forming cells in the embryonic spinal cord were initially NG2(-) and rapidly acquired NG2 in vitro. NG2 and Olig2 were found to be rapidly induced by cell culture conditions in spinal cord neural precursor cells. Olig2 expression was also induced in astrocytes and embryonic peripheral nervous system (PNS) cells in culture after EGF/FGF treatment. These data provide new evidence for profound phenotypic modifications in CNS and PNS neural precursor cells induced by culture conditions.

Comparative expression patterns of T-, N-, E-cadherins, beta-catenin, and polysialic acid neural cell adhesion molecule in rat cochlea during development: implications for the nature of Kölliker's organ.

We investigated the expression patterns of several cell adhesion molecules (CAMs) during rat cochlea ontogeny, from embryo day 16 to adulthood, with the use of immunohistochemistry: neural cadherin (N-cad) and polysialic acid neural CAM (PSA-NCAM) as two different neural CAM paradigms; epithelial cadherin (E-cad), which was restricted to the epitheloid phenotype; and the cytoplasmic domain-free truncated-cadherin (T-cad). We made the following observations. (1) T-cad was present in all types of fibrocyte and in subdomains within the pillar cells. (2) E- and N-cad were expressed with mutually exclusive patterns and did not overlap with T-cad. All cochlear epithelial cells, including the sensory outer hair cells (OHCs), were E-cad-positive, except for the negative inner hair cells (IHCs) and the nonsensory Kölliker's organ domain close to the IHCs. N-cad expression appeared first in the developing IHCs and then in the neighboring Kölliker's organ in an increasingly mediolateral gradient in opposition to the E-cad gradient. The OHCs, which are never N-cad positive, intensively expressed E-cad, as did the Hensen cells at the beginning of their differentiation. (3) The cadherin-linked molecule beta-catenin, absent in fibrocytes, was detected in all epithelial cell membranes and was prominent in the E-cad-rich modiolar extremity of Kölliker's organ. (4) Gradual PSA-NCAM expression was observed in the lateral portion of Kölliker's organ, and the intense PSA-NCAM expression was seen surrounding the IHCs. As development proceeded, PSA-NCAM immunoreactivity progressively became restricted to the basal poles of the IHCs, where it remained in the adult rat cochlea, suggesting a synaptic plasticity. Synaptic plasticity in rat cochlea and hypotheses about T-cad functions and neosensory features of the Kölliker's organ are discussed.

Expression profiles of the connexin genes, Gjb1 and Gjb3, in the developing mouse cochlea.

Several connexin genes (GJB1, GJB2, GJB3, GJB6 and GJA1) have been found mutated in patients with non-syndromic and/or syndromic deafness indicating an important role of these proteins in the auditory system. In order to better understand the function of the connexins in the inner ear we have analyzed the gene expression profiles of two connexin genes, Gjb1 (connexin 32) and Gjb3 (connexin 31), by in situ hybridization during the mouse cochlea organogenesis, from early otocyst up to the mature organ in adult. In the developing otocyst epithelium, some restricted domains expressed Gjb3 and Gjb1 whilst high levels of both transcripts were present in the surrounding mesenchymal tissue. As development proceeds, expression of these two genes was found in various subtypes of fibrocytes, either within the spiral limbus or along the spiral ligament, as well as in the basilar membrane cells, in the Reissner's membrane cells, and in subsets of the cellular elements of the cochlear ganglion. Gjb3 and Gjb1 expression was spatiotemporally modulated within the sensory hair cells and the various supporting cells that compose the developing organ of Corti. A transitory expression of Gjb1 was found in the basal and intermediate cells of the stria vascularis. In the adult cochlea Gjb1 transcripts disappeared while Gjb3 expression remained present in fibrocytes with specific expression patterns.

Expression profiles of the connexin genes, Gjb1 and Gjb3, in the developing mouse cochlea.

Several connexin genes (GJB1, GJB2, GJB3, GJB6 and GJA1) have been found mutated in patients with non-syndromic and/or syndromic deafness indicating an important role of these proteins in the auditory system. In order to better understand the function of the connexins in the inner ear we have analyzed the gene expression profiles of two connexin genes, Gjb1 (connexin 32) and Gjb3 (connexin 31), by in situ hybridization during the mouse cochlea organogenesis, from early otocyst up to the mature organ in adult. In the developing otocyst epithelium, some restricted domains expressed Gjb3 and Gjb1 whilst high levels of both transcripts were present in the surrounding mesenchymal tissue. As development proceeds, expression of these two genes was found in various subtypes of fibrocytes, either within the spiral limbus or along the spiral ligament, as well as in the basilar membrane cells, in the Reissner's membrane cells, and in subsets of the cellular elements of the cochlear ganglion. Gjb3 and Gjb1 expression was spatiotemporally modulated within the sensory hair cells and the various supporting cells that compose the developing organ of Corti. A transitory expression of Gjb1 was found in the basal and intermediate cells of the stria vascularis. In the adult cochlea Gjb1 transcripts disappeared while Gjb3 expression remained present in fibrocytes with specific expression patterns.