Gcm/Glide-dependent conversion into glia depends on neural stem cell age, but not on division, triggering a chromatin signature that is conserved in vertebrate glia

Abstract

SUMMARYNeurons and glia differentiate from multipotent precursors called neural stem cells (NSCs), upon the activation of specific transcription factors. In vitro, it has been shown that NSCs display very plastic features; however, one of the major challenges is to understand the bases of lineage restriction and NSC plasticity in vivo, at the cellular level. We show here that overexpression of the Gcm transcription factor, which controls the glial versus neuronal fate choice, fully and efficiently converts Drosophila NSCs towards the glial fate via an intermediate state. Gcm acts in a dose-dependent and autonomous manner by concomitantly repressing the endogenous program and inducing the glial program in the NSC. Most NSCs divide several times to build the embryonic nervous system and eventually enter quiescence: strikingly, the gliogenic potential of Gcm decreases with time and quiescent NSCs are resistant to fate conversion. Together with the fact that Gcm is able to convert mutant NSCs that cannot divide, this indicates that plasticity depends on temporal cues rather than on the mitotic potential. Finally, NSC plasticity involves specific chromatin modifications. The endogenous glial cells, as well as those induced by Gcm overexpression display low levels of histone 3 lysine 9 acetylation (H3K9ac) and Drosophila CREB-binding protein (dCBP) Histone Acetyl-Transferase (HAT). Moreover, we show that dCBP targets the H3K9 residue and that high levels of dCBP HAT disrupt gliogenesis. Thus, glial differentiation needs low levels of histone acetylation, a feature shared by vertebrate glia, calling for an epigenetic pathway conserved in evolution. KEY WORDS: Neural stem cells, Glia, Drosophila, dCBP, Gcm/Glide, Histone acetylationGcm/Glide-dependent conversion into glia depends on neural stem cell age, but not on division, triggering a chromatin signature that is conserved in vertebrate glia Hakima Flici*, Berra Erkosar*, Orban Komonyi, Omer Faruk Karatas, Pietro Laneve and Angela Giangrande † DEVELOPMENT MATERIALS AND METHODS FliesFlies were raised at 25°C unless otherwise specified. w 1118 was the wild type. Transgenic lines were: UAS-gcm (one dose: F18A: 1XGcm; two doses: M24A: 2XGcm) (Bernardoni et al., 1998); lbe(K)-Gal4, UAS-GFP (Baumgardt et al., 2009); mzVum-Gal4 (Landgraf et al., 2003); apterousGal4, UAS-mRFP (Baumgardt et al., 2007); gcm 34 / CyO,twi-LacZ (Vincent et al., 1996); UAS-dCBP and UAS-dCBP-FLAD (Kumar et al., 2004); repoGal4/TM3 (Sepp et al., 2001); repo 3692 /TM3ubx-lacZ (Halter et al., 1995); repo-Gal4 (Lee and Jones, 2005); and UAS-mCD8GFP, elavGal4, 4 /TM3 (Bloomington Drosophila Stock Center). Immunohistochemistry and in situ hybridizationImmunolabeling and in situ hybridization on embryos were as described previously (Bernardoni et al., 1998). Primary antibodies were: mouse(m)--Repo (1:50), m--Engrailed (1:500) and rat(rt)--Elav (1:200) from DSHB; chicken--GFP (1:1000), rabbit(rb)--RFP (1:500), rb--Caspase3 (1:500), m--H3K9ac (1:500), rb--H3K4me3 (1:500) from Abcam; rb--GFP (1...

Interlocked loops trigger lineage specification and stable fates in the Drosophila nervous system

“…Total RNA was purified using Trizol (Invitrogen), reverse transcribed by SuperScript II and the qPCR was done with the Roche LightCycler480 and Sybr Green Master mix (Roche). Expression levels were automatically calculated (LightCycler480 Software, release 1.5.0) by calibration to gene-specific standard curves generated on input cDNAs 31 . Collected values were normalized to GFP messenger RNA amount, derived from four amplification reactions, each performed on four independent experiments.…”

“…Proteins were extracted from staged embryos from the specified genotypes 31 . A total of 30 mg of protein was separated by 8% SDS-PAGE gel, transferred onto nitrocellulose membrane and probed with primary antibodies diluted in 1 Â PBS, 3% milk: m-a-Flag (1:5,000) (Sigma), ck-a-GFP (1:5,000) (Abcam), m-a-b-gal (1:1,000), m-a-Repo (1:2,000) (DSHB), rb-a-dCBP (1:5,000) (M. Mannervik).…”

“…4j). Since ectopic Gcm works by converting neural stem cells into glia 31 , we analysed the effects of dCBP on this process using the NB marker Deadpan (Dpn). The number of Dpn( þ ) cells decreases on Gcm expression (Fig.…”

Stem cell aging and plasticity in the Drosophila nervous system

“…Of note, the induction of an ambiguous phenotype also characterizes NBs mutant for String challenged with Gcm/ Glide 45 and intermediate fates characterize the majority of cancer cells. 58 We propose that intermediate states represent dynamic features and that cells with altered mitotic programs as those occurring in metastatic processes can express markers of a novel fate but remain blocked in such ambiguous state.…”

“…45 Furthermore, careful inspection of animals overexpressing Gcm/Glide at early stages clearly shows the induction of cells that transiently express the intermediate NB/glial identity before adopting a complete and stable glial fate (reviewed in ref. 45 46 This also seems to be the case in a recently described β-cell regeneration model, in which dying β cells force pancreatic α cells to transdifferentiate and fate conversion. These and more permanent chromatin changes are likely to play central roles in the control of stem cell plasticity.…”