In vivo analysis of quiescent adult neural stem cells responding to Sonic hedgehog
ABSTRACT Sonic hedgehog (Shh) has been implicated in the ongoing neurogenesis in postnatal rodent brains. Here we adopted an in vivo genetic fate-mapping strategy, using Gli1 (GLI-Kruppel family member) as a sensitive readout of Shh activity, to systematically mark and follow the fate of Shh-responding cells in the adult mouse forebrain. We show that initially, only a small population of cells (including both quiescent neural stem cells and transit-amplifying cells) responds to Shh in regions undergoing neurogenesis. This population subsequently expands markedly to continuously provide new neurons in the forebrain. Our study of the behaviour of quiescent neural stem cells provides in vivo evidence that they can self-renew for over a year and generate multiple cell types. Furthermore, we show that the neural stem cell niches in the subventricular zone and dentate gyrus are established sequentially and not until late embryonic stages.
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ABSTRACT: The regenerative capacity of the central nervous system must be optimized to promote repair following traumatic brain injury (TBI) and may differ with the site and form of damage. Sonic hedgehog (Shh) maintains neural stem cells and promotes oligodendrogenesis. We examined whether Shh signaling contributes to neuroblast (doublecortin) or oligodendrocyte progenitor (neural/glial antigen 2 [NG2]) responses in two distinct TBI models. Shh-responsive cells were heritably labeled in vivo using Gli1-CreER(T2);R26-YFP bitransgenic mice with tamoxifen administration on Days 2 and 3 post-TBI. Injury to the cerebral cortex was produced with mild controlled cortical impact. Yellow fluorescent protein (YFP) cells decreased in cortical lesions. Total YFP cells increased in the subventricular zone (SVZ), indicating Shh pathway activation in SVZ cells, including doublecortin-labeled neuroblasts. The alternate TBI model produced traumatic axonal injury in the corpus callosum. YFP cells decreased within the SVZ and were rarely double labeled as NG2 progenitors. NG2 progenitors increased in the cortex, with a similar pattern in the corpus callosum. To further test the potential of NG2 progenitors to respond through Shh signaling, Smoothened agonist was microinjected into the corpus callosum to activate Shh signaling. YFP cells and NG2 progenitors increased in the SVZ but were not double labeled. This result indicates that either direct Smoothened activation in NG2 progenitors does not signal through Gli1 or that Smoothened agonist acts indirectly to increase NG2 progenitors. Therefore, in all conditions, neuroblasts exhibited differential Shh pathway utilization compared with oligodendrocyte progenitors. Notably, cortical versus white matter damage from TBI produced opposite responses of Shh-activated cells within the SVZ.ASN Neuro 07/2014; 6(5). DOI:10.1177/1759091414551782 · 4.44 Impact Factor
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ABSTRACT: eLife digest Neurons that arise in the adult nervous system originate from neural stem cells and neural progenitor cells. Neural stem cells have long lives, much of which they spend in a quiescent state. Neural stem cells can also give rise to neural progenitor cells, which proliferate rapidly during their short lives and then ‘differentiate’ into neurons or glia. Unlike some other tissues, it has not been possible to identify or purify neural stem cells directly from the tissue. Consequently, neural stem and progenitor cells have usually been studied retrospectively, based on their ability to form colonies in laboratory cell cultures. A region of the brain called the subventricular zone contains both neural stem cells and neural progenitor cells, and is one of only two regions of the brain where neural stem cells are found in adult mammals. When cells from the subventricular zone are cultured in a way that allows the cells to freely float around (rather than growing on a surface), a few percent form spherical colonies called neurospheres. Since neurosphere-forming cells can self-renew and differentiate into neurons and glia, the ability of cells to form neurospheres has generally been taken as evidence that they are stem cells. However, the exact relationship between neural stem cells and neurosphere-forming cells has been uncertain. Now, Mich, Signer et al. have used a technique called flow cytometry to identify and isolate neural stem cells and neurosphere-forming cells directly from the subventricular zone. The neural stem cells, which Mich, Signer et al. term pre-GEPCOT cells (based on an acronym of the markers used to isolate the cells), were long-lived and quiescent, but they lacked the ability to form colonies in culture. The neurosphere-forming cells, named GEPCOT cells, were short-lived and highly proliferative in the brain. These results demonstrate that the cells that form neurospheres in culture are not stem cells at all, and that real stem cells are not able to form colonies under existing culture conditions. The identification of undifferentiated pre-GEPCOT and GEPCOT cells will make it possible to directly study the properties of these cells inside the mouse brain, and to isolate live cells to test how they function. The results also highlight the need for new tests to study neural stem cell function, given that current tests using neurospheres do not detect stem cells as commonly assumed. DOI: http://dx.doi.org/10.7554/eLife.02669.002eLife Sciences 05/2014; 3:e02669. DOI:10.7554/eLife.02669 · 8.52 Impact Factor
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ABSTRACT: The hedgehog (Hh) signaling pathway is one of the key regulators in development with a dual role in cell fate specification, proliferation, and survival on different target cells. We have investigated the effect of recombinant sonic hedgehog (r-SHH) on extracted multipotent stem cells from human exfoliated deciduous teeth (SHED), which represent a potential stem cell population for therapeutic applications. Cell proliferation and cycle assays shown that r-SHH did not have a distinctive effect on cell cycle progression, nor did it increase cell number over a wide range of concentrations. Quantitative polymerase chain reaction (Q-PCR) also suggests that r-SHH treatment has no demonstrable influence on expression of proliferative genes (CCNE1 and KI67); in contrast, the anti-proliferative gene (CDKN1A) is overexpressed in response to SHH. Our findings have suggested the possibility that SHEDs demonstrate a different potential from human bone marrow mesenchymal stem cells (h-BMSCs) and dorsal neural progenitor in response to growth factors such as SHH.Cell Biology International 04/2014; 38(4). DOI:10.1002/cbin.10227 · 1.64 Impact Factor