Supplementary MaterialsFigure S1: Dpn protein perdures in mutant (B) 3rd instar larval brain. pone.0046724.s001.pdf (273K) GUID:?13873CEA-A195-4BA7-9DA7-DAB121585F1B Physique S2: Loss of type II NBs in in combination with labels the ABT-737 manufacturer type II NB lineages in a 3rd instar larval brain. Arrows indicate the Ase-negative type II NBs. (BCB) In mutant 3rd instar larval brains, no NBs are labeled by mCD8-GFP driven by with mutant type II NB (arrowhead) at 9C12 hours ALH. Type II NBs were labeled with mCD8-GFP expression (in green) driven by mutant (BCB) brain lobe at embryonic stage 14/15 (10.5C13.5 hrs AEL) is stained with Ase in red and Dpn in green. Arrows point to Dpn+Ase- NBs. Wild type and mutant brains contain similar numbers of Dpn+Ase- NBs.(PDF) pone.0046724.s004.pdf (313K) GUID:?F7B93A2E-1D38-45E1-B26C-50A2495021C4 Physique S5: mutant MB clones show various degrees of loss of late-born neurons. Among total 25 mutant clones examined, 17 clones show the loss of late-born / neurons as indicated by axon reduction in the center of the / lobe shown in a single focal plane (DCD), 3 clones show a complete loss of / neurons (E), and 5 clones have only neurons (F). Insets in (C) and (F) show the cell body regions (outlined by dashed circles) of the corresponding clones. Note that the MB clones with only neurons contain much fewer MB neurons compared to wild type clones.(PDF) pone.0046724.s005.pdf (110K) GUID:?2CC4CDCF-2EF2-4CB2-85C9-8E7CF3E0CE78 Figure S6: (A) and double mutant (B) type I NB clones in adult brains. Clones were labeled with mCD8 in red and stained for Dpn in green and Ase in blue. Numerous type I NBs are present in individual clones.(PDF) pone.0046724.s006.pdf (64K) GUID:?E3F5EE15-4CB1-4137-882B-0E891317EF3E Physique S7: Dpn overexpression in MARCM clones results in overproliferation in both type I and type II NB lineages. (A) A wild type type II NB clone has a single Ase-negative ABT-737 manufacturer NB (arrow) and a couple of Ase-negative immature INPs (arrowheads). (B) A type II NB clone overexpressing Dpn contains multiple NBs (arrows) and numerous immature INPs (arrowheads). (C) A wild type type I NB clone contains a single Ase-positive NB and a few Ase-positive GMCs IKBKB antibody (arrowheads). (DCD) A type I NB clone overexpressing Dpn has a single NB (arrow) but an ABT-737 manufacturer increased number of GMCs (arrowheads). (ECE) A type I NB clone overexpressing Dpn contains multiple NBs (arrows) and increased number of GMCs.(PDF) pone.0046724.s007.pdf (100K) GUID:?23CA26DF-C8E2-4200-9890-12BEA456B337 Figure S8: Ectopic Dpn expression causes immature INPs to become mitotically active. (ACA) phospho-histone H3 (p-H3) is not detected in Ase-negative immature INPs (arrows) in wild type type II NB lineages. (BCB) Anti-p-H3 labels many immature INPs (arrows) when Dpn is usually ectopically expressed. Type II NB lineages are labeled by mCD8-GFP driven by combined with also drives the expression of FLPase, which excises the stop codon and results in the expression of -gal under the control of the constitutively active promoter. Through this approach, -gal is expressed in type II NBs (arrows), which lack Ase, as well as type II NB progeny, which are also labeled by mCD8-GFP. (BCB) In brains expressing Notch-RNAi, there is very little mCD8-GFP expression and there are no -gal+ NBs, indicating Notch is necessary for maintaining NBs.(PDF) pone.0046724.s009.pdf (343K) GUID:?F22B5ADF-F505-495C-843D-AD360A2E644A Abstract Neural stem cells (NSCs) are able to self-renew while giving rise to neurons and glia that comprise a functional nervous system. However, how NSC self-renewal is usually maintained is not well comprehended. Using the larval NSCs called neuroblasts (NBs) as a model, we demonstrate that this Hairy and Enhancer-of-Split (Hes) family protein Deadpan (Dpn) plays important roles in NB self-renewal and specification. The loss of Dpn leads to the premature loss of NBs and truncated NB lineages, a process likely mediated by the homeobox protein Prospero (Pros). Conversely, ectopic/over-expression of Dpn promotes ectopic self-renewing divisions and maintains NB self-renewal into adulthood. In type II NBs, which generate transit amplifying intermediate neural progenitors (INPs) like mammalian NSCs, the loss of Dpn results in ectopic expression of type I NB markers Asense (Ase) and Pros before these type II NBs are lost at early larval stages. Our results also show that knockdown of Notch leads to ectopic Ase expression in type II NBs and the premature loss of type II NBs. Significantly, expression is usually unchanged in these transformed NBs. Furthermore, the loss of Dpn does.