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  • br Materials and methods br Statistical analysis Data are pr

    2018-10-24


    Materials and methods
    Statistical analysis Data are presented as mean±SEM. Differences between groups were detected for statistical significance using the unpaired Student\'s t-test. P<0.05 was considered significant.
    Results
    Discussion We have previously postulated that Hopx might label NSCs in the dentate gyrus based upon its co-expression with GFAP in the SGZ (De Toni et al., 2008). Here we demonstrate that Hopx in the SGZ of the DG co-localizes with NSC markers including GFAP, Sox2 and Nestin, but not with transit-amplifying cell markers Mash1 or Tbr2. Our lineage tracing experiments demonstrate that Hopx+cells in the adult DG give rise to DCX-expressing neuroblasts and then granule neurons over time, confirming that these Hopx+cells are indeed NSCs. Collectively, these data establish that Hopx is a novel marker of quiescent NSCs in the adult DG. Hopx also plays a functional role in NSCs in the adult hippocampus. Our previous work has shown that neuronal production is increased in Hopx null hippocampi (De Toni et al., 2008). This is consistent with our current findings demonstrating that neurogenesis is enhanced in Hopx null DGs, whereas quiescent NSCs are reduced in number. This finding suggests that Hopx modulates neurogenesis and NSC self-renewal. Of note, our current data were quantified using anatomically matched histological sections. Future stereological analyses of Hopx heterozygous and Hopx null DGs will allow for more definitive measures of cell numbers and structural volumes between genotypes (West, 1999; Lagace et al., 2007; Gao et al., 2007). Clearly, however, there are additional and alternative pathways regulating these processes since the phenotypes we observe are relatively mild, and some degree of NSC self-renewal persists in null animals. Indeed, many signaling pathways have been implicated in adult neurogenesis, including Notch, Wnt, Bmp, and Shh (Faigle & Song, 2013). Notch signaling plays a particularly important role in the maintenance of quiescent NSCs. Genetic SCR7 of Notch signaling components including Jagged1, Notch1, and Rbp-J, or exposure to γ-secretase inhibitors, disturb the balance between NSC maintenance and sustained neurogenesis (Giachino & Taylor, 2014; Hitoshi et al., 2002; Lavado & Oliver, 2014; Breunig et al., 2007). Notch signaling is down-regulated in the Hopx null DGs and decreased Notch activation likely contributes to the neurogenic phenotypes that we have described. Alternatively, the reduction of Hey2 may be attributed to Notch-independent pathways such as FGF, in which Hopx might serve as an intermediate (Doetzlhofer et al., 2009; Al Alam et al., 2015). Future studies will focus on how Hopx functions to regulate Notch and NICD activation, and whether this pathway is relevant to Hopx function in other adult stem cell populations in multiple organs and tissues. Future studies will also focus on differential analyses of dorsal and ventral hippocampi at both the histological and gene expression levels that could reveal additional differences between Hopx mutants and controls.
    Conclusion
    Acknowledgments We thank Epstein Lab members, Dr. Russell C. Addis and Dr. Peter S. Klein for their helpful discussions and assistance, the Penn Cardiovascular Institute Histology Core for technical assistance, and the CDB Microscopy Core for confocal imaging. We also thank Dr. Hyung Song Nam, University of Utah, for advice on the manuscript. The work was supported by NIHU01 HL100405, R01 HL071546, the Cotswold Foundation and the Spain Fund to J.A.E, and R01 MH066912 to S.A.A.
    Introduction Acute myeloid leukemia (AML) is a heterogeneous malignancy characterized by bone marrow infiltration of immature leukemia blasts (Network, 2013; Welch et al., 2012; Reikvam et al., 2013a). The AML cell population has a hierarchical organization including a minority of leukemic stem cells (Bonnet & Dick, 1997) that often are included in the CD34+/CD38− compartment (Bonnet & Dick, 1997). These cells show self-renewal and long-term in vitro proliferation and have the capacity to produce leukemic progenitors showing proliferative but not self-renewal capacity (Huntly & Gilliland, 2005). Both leukemic as well as normal hematopoietic stem cells (HSCs) depend on support from the bone marrow microenvironment (Lane et al., 2009). This support is at least partly mediated through the local cytokine network and the leukemic progenitors (i.e. the more mature leukemia cell subset) as well as the stromal cells contribute to this network through their constitutive cytokine release.