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Conceived and designed the experiments: SZ JW SB SY YH TL. Performed the experiments: SZ JW SB SY YH. Analyzed the data: SZ JW SB SY YH. Contributed reagents/materials/analysis tools: SZ JW SB SY YH TL. Wrote the paper: SZ JW SB SY YH TL.
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 maintained is not well understood. Using the Drosophila larval NSCs called neuroblasts (NBs) as a model, we demonstrate that the 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, dpn expression is unchanged in these transformed NBs. Furthermore, the loss of Dpn does not inhibit the over-proliferation of type II NBs and immature INPs caused by over-expression of activated Notch. Our data suggest that Dpn plays important roles in maintaining NB self-renewal and specification of type II NBs in larval brains and that Dpn and Notch function independently in regulating type II NB proliferation and specification.
Neural stem cells (NSCs), like other stem cells, maintain their undifferentiated proliferative status while undergoing many rounds of cell division to produce a diverse array of neurons and glia. Proliferating NSCs are maintained through symmetric divisions, which expand the NSC pool, as well as self-renewing asymmetric cell divisions, which produce one daughter that becomes a NSC and another daughter with limited proliferative potential that will produce differentiated progeny. Maintaining the self-renewal of NSCs is critical for the proper formation and homeostasis of the nervous system. Premature termination of NSC self-renewal can lead to the reduction or loss of particular cell types, whereas continued or increased NSC self-renewal can lead to tumor formation. Hence, deciphering the mechanisms underlying the balance between NSC self-renewal and neuronal differentiation is important for understanding normal neurogenesis as well as the pathology of diseases that result from perturbations in NSC self-renewal. However, the mechanisms maintaining NSC self-renewal and how NSC proliferation and differentiation are balanced are just beginning to be understood.
Drosophila larval NSCs (called neuroblasts, or NBs) are an excellent simple model system for studying the basic, conserved biology of NSCs. In the developing fly central brain, there are two types of NBs (type I and type II). These fly NBs, especially the newly identified type II NBs (also called posterior Asense-negative [PAN] or Dorsomedial [DM] NBs), are analogous to mammalian radial glial cells, which function as NSCs in the developing mammalian brain [1]. The type II NBs produce an intermediate neural progenitor (INP) that undergoes several self-renewing divisions to amplify the number of progeny produced by each NB (Fig. 1A) [2], [3], [4]. Thus, fly INPs are comparable to the mammalian intermediate progenitor, particularly the self-renewing outer-subventricular zone radial glia-like (oRG) cells that amplify the number of progeny descended from radial glial cells as reported in human embryos and ferrets [5], [6], [7]. In each round of self-renewing divisions, individual INPs produce a ganglion mother cell (GMC), which divides once to produce two neurons. The developing fly brain, however, is mostly populated by type I NBs that give rise directly to GMCs instead of self-renewing INPs (Fig. 1A). Drosophila larval NBs are derived from embryonic NBs. After several rounds of self-renewing divisions, most embryonic NBs become mitotically quiescent at late embryonic stages, except for the four mushroom body NBs and a basal anterior NB. These quiescent NBs re-enter the cell cycle during 1st and 2nd instar larval stages and undergo repeated self-renewing divisions until early pupal stages [8], [9].
Studies of Drosophila NBs have identified a number of genes that regulate NB self-renewal. The majority of these genes, such as aPKC, Partner of inscuteable (Pins), the NuMA-related Mushroom body defect (Mud), aurora-A, polo, and protein phosphatase 2A (PP2A), are involved in asymmetric cell division (reviewed in [10], [11]. These genes ensure the proper segregation to a single daughter cell of cell fate determinants, such as Prospero (Pros), Numb, and Brat. These cell fate determinants promote cell cycle exit of GMCs or maturation of INPs [4], [12], [13]. Defects in asymmetric cell division or loss of cell fate determinants perturb the normal pattern of NB self-renewal, leading to an increased number of NBs or, conversely, differentiation. In the type II neuroblast lineage, the self-renewal potential of INPs is limited by the transcription factor Earmuff (Erm), which positively regulates pros expression [14]. However, the proteins that act within NBs to promote NB self-renewal remain largely unknown.
To understand how the self-renewal of NBs is regulated, we investigated the function of the Drosophila bHLH transcriptional repressor Deadpan (Dpn), a member of the Hairy and Enhancer-of-Split (Hes) family that is expressed in all neural precursors [15]. We reasoned that genes that are required for maintaining NBs might be specifically expressed in NBs, but not in their progeny. Dpn is specifically expressed in all the larval NBs as well as in the self-renewing INPs [3], [4], [15], making Dpn a good candidate for maintaining NB self-renewal. Our work demonstrates that Dpn is both necessary and sufficient for maintaining NB self-renewal. Furthermore, we show that Dpn and Notch function in separable pathways to suppress Ase expression in type II NBs and maintain the self-renewal of type II NBs.
The following fly stocks were used for dpn loss-of-function analyses: dpn1, dpn7, and Df(2R)Exel7095. Other fly stocks include: FRT42D dpn7/CyO; FRT42D dpn1/CyO; and hs-Flpase elav-Gal4 UAS-mCD8-GFP; FRT42D tub-Gal80 for generating dpn mutant clones; ase-Gal4, insc-Gal4 (Gal41407 inserted in inscuteable promoter), UAS-dpn for ectopic/over-expression of Dpn; and Gal414–94 [16] for transgene expression in type II NBs; UAS-FLPase and actin-FRT-Stop-FRT-lacZ for lineage tracing of type II NBs; ase-Gal80 transgenic lines were generated as described below to restrict the expression of insc-Gal4 to the type II lineages.
The defined ase promoter (1.6 kb upstream of the transcription start site) together with 455 bp 5′untranslated region of the ase transcription unit [17] was amplified by PCR and subcloned into pCaSpeR4 between EcoRI and NotI. The SV40 poly(A) tail and the Gal80 coding region were then sequentially subcloned into XbaI–StuI and NotI–XbaI sites, respectively, downstream of the ase promoter in pCaSpeR4 to generate ase-Gal80.
dpn mutant MARCM [18] clones were induced by 1 hour heat shock at 38°C at 0 day or 1 day after larval hatching. Brains were dissected at various developmental stages as indicated for the examination of clonal phenotypes.
Larval or adult brains were dissected, fixed, and stained as described before [19]. Primary antibodies used in this study include: rabbit anti-Mira (1500), guinea pig anti-Ase (15000), rat anti-mCD8 (Caltag, 1100), 1D4 mAb (Hybridoma Bank, 150), mouse anti-Pros (Hybridoma Bank, 120), rabbit anti-Dpn (1500). Secondary antibodies conjugated to Cy2, Rhodamine Red X, or Cy5 (Jackson ImmunoResearch, West Grove, PA, USA) were used at 1100, 1500, or 150, respectively. Alexa Fluor 488 Phalloidin (Invitrogen Carlsbad, CA, USA) was used at 140. Images were taken with Leica SP2 and SP5 confocal microscope and processed with Adobe Photoshop. Two-tailed t-tests were used for statistical analyses.
There are approximately 100 NBs in the central brain of each Drosophila larval brain lobe. The majority of these NBs are specified during embryonic stages. After a period of quiescence during late embryonic and early larval stages, these NBs are reactivated and proliferate until around 1 day after pupal formation (APF), at which point they undergo terminal divisions [8]. An exception to this pattern are the mushroom body NBs, which proliferate continuously from embryonic stages until the end of pupal stages [9]. We predict that the loss of any gene necessary for NB maintenance will result in a premature disappearance of NBs, and thus fewer NBs during larval stages. To examine whether Dpn is required for maintaining NB self-renewal, we first compared the number of type I and type II NBs in wild type and dpn mutant brains at the late 3rd instar larval stage (4 days after larval hatching [ALH]) using Miranda (Mira), which marks all larval NBs, and Asense (Ase), which is expressed in type I but not type II NBs. Three dpn alleles, including the null allele dpn1, the dominant negative allele dpn7, and the deficiency line Df(2R)Exel7095 that removes the entire dpn gene, were used to examine dpn mutant phenotypes (note that the dpn7 allele produces a dominant-negative Dpn protein that is still recognized by the anti-Dpn antibody). Each brain lobe in a wild type 3rd instar larva normally contains eight type II NBs on the dorsal side (Fig. 1B) and approximately 90 type I NBs located on both dorsal and ventral sides (Fig. 1D). Strikingly, type II NBs were completely absent in dpn1/dpn7 mutant brains (Fig. 1C). The number of type I NBs was also significantly reduced to 45–65 type I NBs per brain lobe in dpn1/dpn7 mutants (Fig. 1E, F). Similar results were observed in dpn1/Df(2R)Exel7095 and dpn7/Df(2R)Exel7095 transheterozygous larval brains (data not shown). These results are consistent with the results recently reported by San-Juan and Baonza (2011), and Zacharious et al. ([20], [21].
The complete loss of type II NBs in 3rd instar dpn mutant brains could reflect (1) a premature disappearance of NBs during embryonic or early larval stages, (2) an inability to generate NBs during embryogenesis, (3) a transformation of type II NBs into type I NBs, or (4) a failure to exit quiescence at early larval stages. One way to distinguish these possibilities is to remove Dpn after type II NBs are formed by generating dpn mutant clones. Although Dpn is absent in dpn1 whole mutant animals (Fig. S1A–B'), Dpn protein perdures in dpn1 clones. When dpn1 clones were induced at 1 day ALH and examined 2 days later, Dpn was still detected in both type I and type II NBs, albeit at much lower levels than in neighboring dpn1 heterozygous NB lineages (Fig. S1C, C', E, E'). Only 4 days after clone induction was Dpn reduced to undetectable levels in dpn1 mutant clones (Fig. S1D, D', F, F'). The perdurance of Dpn in dpn mutant clones made it difficult to unambiguiously determine the role of Dpn in larval NBs (except for MB NBs, see below) by clonal analyses.
To investigate when type II NBs are lost in dpn mutant brains, we first characterized the development of type II NB lineages in wild type 1st instar larvae and looked for type II NBs in dpn1/dpn7 mutants at the same stage. To specifically label type II NBs and their progeny, we used UAS-mCD8-GFP driven by a combination of inscuteable (insc)-Gal4 [22] and asense (ase)-Gal80. Insc-Gal4 is active in all NBs whereas ase-Gal80 is expressed only in type I NBs. Therefore, insc-Gal4 in combination with ase-Gal80 restricts the expression of mCD8-GFP to type II NB lineages (Fig. 2A, also see Fig. S2A, A'). Using this approach, the earliest time point at which we were able to definitively identify all 8 type II NBs in wild type brains was 9–12 hours ALH, when the type II NBs have likely just exited quiescence (Fig. 2B–C"). In wild type 1st instar larvae, type II NBs and their progeny are clustered into 3 groups in each brain lobe: two groups, comprised of 3 NBs each, are positioned on the medial side of the brain and 2 NBs are clustered on the lateral side (Fig. 2A'). Consistent with the notion that type II NBs were just emerging from quiescence at this time, there were relatively few INPs and GMCs at 9–12 hours ALH (Fig. 2C–C"). At 18–24 hours ALH, each brain lobe contained increased numbers of type II NB progeny, including mature INPs and GMCs (Fig. 2D–D").
In dpn1/dpn7 brain lobes at 9–12 hours ALH, we observed 3 clusters of mCD8-GFP+ cells in each brain lobe, but only identified on average 2 mCD8-GFP-labeled NBs per brain lobe (Fig. 2B, E–E"). After 9–12 hours ALH, we rarely observed type II NBs in the mCD8-GFP+ clusters in dpn1/dpn7 brains, except for some small Ase+ Dpn+ cells resembling INPs (Fig. 2B, F, F', also see Fig. S2B, B'). Interestingly, unlike normal type II NBs, which do not express Ase and Pros, the mCD8-GFP-labeled dpn mutant NBs observed at 9–12 hours ALH ectopically expressed Ase and nuclear Pros, making type II NBs appear similar to type I NBs at the same stage (Fig. 2E–E', Fig. S3). To investigate whether type II NBs are generated and specified correctly, we examined type II NBs in wild type and dpn mutant brains at embryonic stage 14/15 (10.5–13 hours after egg laying [AEL]). Interestingly, unlike larval brains, which contain only 8 Dpn+ Ase- NBs per hemisphere, embryonic brains contain 33.2±4 (n=6) Dpn+Ase- NBs per hemisphere at stage 14/15 (Fig. S4A–A"). A similar number (32.3±4.8, n=6) of Dpn+Ase- NBs were also observed in dpn mutant brains at the same stage (Fig. S4B–B"). Although it is not clear which of these embryonic Dpn+Ase- NBs become larval type II NBs, these results indicate that the loss of Dpn likely does not affect the generation and specification of type II NBs at embryonic stages. The ectopic expression of Ase and Pros in dpn mutant type II NBs during early larval stages suggests that Dpn may be required for maintaining their proper identity of type II NBs only after they exit quiescence, but not earlier during embryonic stages. In the absence of Dpn, type II NBs are transformed into type I-like NBs after they exit quiescence and may eventually lose the expression of mCD8-GFP driven by insc-Gal4 ase-Gal80 due to the activation of the ase promoter and subsequent expression of Gal80. However, since no ectopic GFP- type I NBs were found at locations where type II NBs reside (Fig. 2F, F', F"), it is likely that these transformed dpn mutant type II NBs were lost prematurely due to a defect in maintaining self-renewal soon after they exit quiescence.
To investigate whether the reduction of type I NBs in 3rd instar dpn mutant brains is due to either a premature loss or a defect in NB formation, we next examined at different developmental stages the well-characterized type I NB lineages that produce the two bilaterally symmetric mushroom bodies (MBs) in the adult fly brain. A single MB is derived from four NBs, each of which divides repeatedly from late embryonic to late pupal stages to sequentially generate three distinct types of MB neurons (γ, α'/β', and α/β) (Fig. S5A, B) [19]. To analyze the MB lineage, we used insc-Gal4, which labels all larval NBs, to drive the expression of mCD8-GFP. UAS-mCD8-GFP driven by Insc-Gal4 labels not only MB NBs, but also the newly born MB neurons and their neurites, which form a distinctive structure. By tracing the neurite bundles from the MB dendritic calyces back to their cell bodies it is possible to identify MB NBs (Fig. 3A, B). In wild type animals, each brain lobe has 4 MB NBs until 2 days APF (Fig. 3A–B', E). However, in dpn1/dpn7 mutants, MB NBs were progressively lost. At the late 3rd instar larval stage, nearly half of MB NBs were missing in dpn mutant brains (Fig. 3C, C', E) and by the start of pupariation, 90% of MB NBs were lost (Fig. 3E). By 1 day APF, no MB NBs were found (Fig. 3D, E). Premature progressive loss of dpn mutant MB NBs suggests that Dpn is required to maintain MB NB self-renewal.
In order to assess whether Dpn functions cell-autonomously to maintain MB NB self-renewal, we next examined dpn loss-of-function phenotypes in MB NB clones that were generated right after larval hatching. The MB NBs keep dividing till the end of pupal stage and produce the longest lineages in the fly brain. Given that Dpn perdures in dpn mutant clones for at least 2 days after clone induction, we examined the phenotype in MB NB clones at 1–2d APF, which is about 5–6 days after clone induction. At 1–2d APF, wild type MB NB clones always contained a NB (n=10) (Fig.3F–F'). In contrast, most (7/8) dpn7 mutant MB NB clones had lost their NBs at 1–2d APF (Fig. 3G–G'). As a result, dpn mutant MB NBs failed to produce late-born MB neurons, resulting in truncated MB lineages that contained fewer MB neurons in adults (Fig. S5C–F). The premature loss of the MB NBs in dpn mutant clones suggests that Dpn functions cell-autonomously to maintain MB NB self-renewal.
To determine whether the loss of other type I NBs in dpn mutant larval brains is similarly due to a defect in maintaining NB self-renewal, we counted the number of type I NBs in wild type and dpn mutant larval brains at different developmental stages. In Drosophila larval brains, NBs start to exit quiescence after 8 hours ALH. By the end of the second instar larval stage, the majority of NBs have exited quiescence and remain proliferative until 1d APF [9]. We compared the number of type I NBs in wild type and dpn mutant larval brains at 9–12, 48, and 96 hours ALH. At 9–12 hours ALH, although most NBs were still quiescent and very small, we were able to identify 94±6.7 type I NBs in wild type brains based on Dpn and Ase staining. The number of type I NBs remained similar at 48 and 96 hour ALH in wild type animals (Fig. 1F, data not shown). In dpn mutant larval brains, the number of type I NBs was close to that in wild type animals at 9–12 hours ALH (87.8±6.7), but was reduced to 64.2±7.1 at 48 hours ALH and remained stable from 48 hours ALH to 96 hours ALH (Fig. 1F). Therefore, the loss of non-MB type I NBs in dpn mutant brains is also due to a defect in maintaining NB self-renewal, but this loss of NBs mainly occurred within 48 hours ALH.
The premature loss of NBs in dpn mutant brains could be due to either apoptosis or precocious terminal division. Apoptosis is unlikely the cause of NB loss since activated caspase was not detected in dpn mutant NBs and the ectopic expression of the caspase inhibitor p35 failed to rescue NB loss (data not shown). In developing fly brains, the majority of NBs (except the MB NBs) undergo a terminal division around 1 day APF [9]. Previous work revealed that the terminal division of most central brain NBs is correlated with the nuclear accumulation of the transcription factor Pros, which promotes cell cycle exit [12], [13]. To test the possibility that Pros prematurely accumulates in the nuclei of dpn mutant NBs and causes their terminal divisions, we first examined whether Pros precociously accumulates in the nuclei of dpn mutant type I NBs at the 3rd instar larval stage. In type I NBs of wild type 3rd instar larvae, only cortical Pros was detected (Fig. 4A–A"). In contrast, Pros was present in the nuclei of multiple type I NBs in 3rd instar dpn1/dpn7 brains (Fig. 4B–B"), suggesting that dpn mutant type I NBs undergo Pros-mediated premature terminal division.
To further determine whether Pros mediates the premature terminal division of dpn mutant NBs, we tested (1) whether the loss of Pros would prevent the premature loss of dpn mutant NBs by generating pros loss-of-function (prosv17) clones in dpn1/dpn7 mutant brains, and (2) whether reducing Pros amounts could partially rescue the loss of type I NBs in dpn mutant larval brains. Consistent with previous reports [4], [23], prosv17 type I NB clones encompassed multiple Ase+ NBs (Fig. 4D, compared with the wild type clone in Fig. 4C). prosv17 mutant type I NB clones generated in dpn1/dpn7 brains had a similar number of NBs (Fig. 4E) as those generated in wild type brains at the 3rd larval stage (13.7±2.1 NBs/clone in dpn1/dpn7 brains, n=3; 14.3±3.8 of NBs/clone in wild type brains, n=3). Furthermore, these pros dpn double mutant type I NBs were maintained through adulthood like pros mutant type I NBs (Fig. S6A–B), whereas a wild type adult brain does not contain any NBs (see below). Consistently, when pros expression was reduced by removing one wild type copy of pros, the number of dpn mutant type I NBs increased by about 15% (Fig. 4F). These data suggest that the premature terminal division of dpn mutant type I NBs is likely mediated by Pros. However, we did not find any prosv17 mutant clones with Ase- type II NBs in dpn1/dpn7 brains (data not shown). Since Ase is ectopically expressed in dpn mutant type II NBs as early as the 1st instar larval stage (Fig. 2E–E"), it is possible that Ase remained mis-expressed in prosv17 type II NB clones in dpn1/dpn7 mutant 3rd instar larvae. The ectopic expression of Ase makes it difficult to conclude whether dpn mutant type II NBs are maintained in the absence of Pros.
Having found that dpn is necessary for maintaining NB self-renewal, we next asked whether Dpn is sufficient to promote NB self-renewal by testing whether the ectopic expression of Dpn in non-self-renewing cells such as immature INPs or GMCs results in ectopic self-renewal, and whether over-expressing Dpn in NBs results in abnormally prolonged self-renewal. If Dpn is sufficient to promote self-renewal, the ectopic expression of Dpn in non-dividing immature INPs and terminally dividing GMCs should lead to ectopic proliferation and increased numbers of immature INPs and GMCs in type II and type I NB lineages. The ectopically proliferating immature INPs and GMCs may even adopt a NB-like fate, leading to increased numbers of type II and type I NBs. Furthermore, Dpn ectopic/over-expression may prolong the maintenance of NBs into adulthood. Since no immature INP- or GMC-specific Gal4 lines are available, we used insc-Gal4 ase-Gal80 for Dpn ectopic/over-expression in type II NB lineages, and ase-Gal4 [24] or insc-Gal4 for Dpn expression in type I NB lineages. In type II lineages, UAS-CD8-GFP driven by insc-Gal4 ase-Gal80 was expressed not only in the NBs but also in INPs and GMCs (Fig. 5A). Similarly, in the type I NB lineage, UAS-CD8-GFP driven by ase-Gal4 or insc-Gal4 was expressed in both NBs and GMCs (Fig. 5C and data not shown). Therefore, these drivers allowed us to over/ectopically express Dpn in NBs as well as immature INPs and GMCs.
We first examined how Dpn ectopic/over-expression affected type II NB lineage development. Strikingly, ectopic/over-expression of Dpn resulted in a massive increase in the number of type II NBs (131±15 type II NBs per brain lobe, Fig. 5B, B') and numerous small Ase- immature INPs (Fig. 5B) in 3rd instar larval brains, compared to 8 type II NBs and 2–3 immature INPs in each type II NB lineage in wild type brains (Fig. 5A). Moreover, the ectopic type II NBs and immature INPs resulting from Dpn ectopic/over-expression even existed in adult brains (Fig. 6B, B'), which normally do not have any NBs or INPs (Fig. 6A, A') [9], [13], [25]. Similarly, when Dpn was ectopically/over-expressed in type II NB clones using insc-Gal4, the clones were much larger and contained more NBs and immature INPs than wild type clones (11.8±3.1 type II NBs and 94.6±13 immature INPs in clones over-expressing Dpn, n=5; control type II NB clones had only a single NB and 2.3±0.5 immature INPs, n=6) (see Fig. S7A, B). The ratio of immature INPs to NBs also increased from 21 to 81, suggesting that Dpn over-expression mainly promotes the over-proliferation of immature INPs. As a result, clones over-expressing Dpn contained relatively fewer mature INPs and neurons per NB than control clones (4.6±1.4 mature INPs/NB and 7.9±1.5neurons/NB in clones over-expressing Dpn, n=5; 19.3±8 mature INPs/NB and 45.4±10.7 neurons/NB in wild type clones, n=6), indicating that the proliferating immature INPs mainly produced immature INPs and/or type II NBs rather than giving rise to mature INPs and neurons.
To confirm that immature INPs indeed undergo ectopic self-renewing divisions in response to Dpn ectopic/over-expression, we examined the expression of a mitotic marker phospho-histone H3 (p-H3) and Mira in these immature INPs. Immature INPs normally do not divide and are negative for p-H3 (Fig. S8A, A'). However, in larval brains over/ectopically expressing Dpn, many immature INPs were ectopically labeled by p-H3 (Fig. S8B, B'), indicating that they were actively dividing. Moreover, Mira formed a crescent in some of these mitotic immature INPs (insets in Fig. 5B, B', compared to the even distribution of Mira on the membrane of wild type immature INPs as shown in insets in Fig. 5A, A'). This asymmetric localization pattern is reminiscent of Mira distribution in NBs (Fig. 5B, B'), indicating that these immature INPs divided asymmetrically. The ectopic self-renewing division of immature INPs and the prolonged existence of type II NBs in adult brains resulting from Dpn ectopic/over-expression provide strong evidence that Dpn is sufficient to promote NB self-renewal.
Similar to the overproliferation phenotypes observed in type II NB lineages, ectopic/over-expression of Dpn driven by ase-Gal4 or insc-Gal4 (data not shown) in type I NB lineages also resulted in an increased number of NBs and GMCs (Fig. 5D–D', compared to Fig. 5C, C'; also see Fig. S7C–E'), some of which even persisted in adult brains (Fig. 6C, C'). Furthermore, Mira also formed a crescent in some GMCs (insets in Fig. 5D, D'), as in the case of immature INPs upon Dpn over-expression. These observations suggest that ectopic/over-expression of Dpn is sufficient to cause terminally dividing GMCs to undergo self-renewing asymmetric divisions. Some of these ectopically self-renewing GMCs may eventually enlarge and become NBs, resulting in an increased number of type I NBs. However, compared to the over-proliferation phenotypes observed in type II NB lineages, the over-proliferation of type I NBs and GMCs in response to ectopic/over-expression of Dpn in type I NB lineages is milder. Dpn ectopic/over-expression in type I NB clones only increased the number of type I NBs by 2 fold and GMCs by 3 fold (1.7±1.5 NBs and 18.2±19.7 GMCs in clones over-expressing Dpn, n=11; 1 NB and 5.7±1.7 GMCs in wild type clones, n=11), whereas Dpn ectopic/over-expression in type II NB clones resulted in a more than 10-fold increase in the number of type II NBs and an over 50-fold increase in the number of immature INPs. Furthermore, ectopic type I NBs resulting from Dpn ectopic/over-expression only persisted in half of adult brain examined (n=15) (Fig. 6C, C'), whereas ectopic type II NBs existed in every adult brains (n=15) (Fig. 6B, B').
Taken together, Dpn ectopic/over-expression phenotypes in both larval and adult brains suggest that Dpn is sufficient to promote the self-renewal of both NB types, rather than just type II NBs as reported by San-Juan and Baonza [21]. However, type II NB lineages are more susceptible than type I NB lineages to the increase in Dpn expression levels.
Drosophila Hes family proteins, such as Hairy and Enhancer-of-split, are known to mediate Notch signaling [26]. Upon activation of the Notch receptor, the intracellular domain of Notch (NIC) translocates to the nucleus and activates the transcription of Hes family proteins. In Drosophila, Notch is expressed in larval NBs [27]. Decreasing Notch activity, either by RNAi knockdown or by expressing Numb, largely eliminates type II NBs, whereas increasing Notch activity by expressing the intracellular domain of Notch (NIC) results in a dramatic increase in NB numbers, particularly the type II NBs [4], [28], [29]. These phenotypes are similar to those caused by manipulating Dpn levels in type II NBs. Therefore, we asked whether Dpn functions downstream of Notch in type II NBs. To address this question, we first examined Dpn expression at different developmental stages when Notch is knocked down in type II NBs using Notch RNAi driven by Gal414–94, which is expressed in type II NBs and INPs but not in type I NBs lineages (Fig. 7A-A", Fig. S9A-A") [16]. Knockdown of Notch leads to the progressive loss of type II NBs labeled by mCD-GFP driven by Gal414–94. At 48 hours ALH, we observed 6.3±1.1 (n=7) type II NBs per brain lobe (Fig. 7B–B"). The number of type II NBs was further reduced to 1.8±0.8 per brain lobe (n=6) (Fig. 7C–C") at 54–72 hours ALH. At 96 hours ALH, no type II NBs were observed (n=8) (Fig. S8B–B"). However, the amount of Dpn in the remaining type II NBs at 48 or 54–72 hours ALH stayed similar to neighboring type I NBs, suggesting that the knockdown of Notch does not affect the expression of Dpn in type II NBs. Interestingly, we found that the knockdown of Notch led to the ectopic expression of the type I NB marker Ase in type II NBs (Fig. 7B–C"). Furthermore, Dpn+Ase+ INPs were largely eliminated at 54–72 hours ALH in the remaining type II NBs lineages. Instead, only a few Ase+ GMC-like cells were observed beside the remaining type II NBs, making the type II NB lineages appear as type I-like NB lineages (Fig. 7C–C"). The ectopic expression of Ase in type II NBs led us to wonder whether the progressive loss of type II NBs resulting from the knockdown of Notch is due to a transformation of type II NBs into type I NBs. We next performed a lineage tracing experiment using UAS-FLPase and actin-FRT-Stop-FRT-lacZ. The expression of FLPase driven by GAL414–94 in type II NBs and their progeny will lead to the excision of the stop codon in actin-FRT-Stop-FRT-lacZ and subsequent expression of lacZ under the control of the actin promoter (Fig. S9A–A"). Since the actin promoter is constitutively active, this approach allows us to label type II NB lineages with lacZ independent of type II NB-specific driver at late larval stages. Using this approach, we labeled the progeny derived from type II NBs with lacZ at 96 hours ALH when Notch was knocked down. However, we did not observe any NBs expressing lacZ, suggesting that knocking down Notch results in a defect in the maintenance of type II NB self-renewal. Therefore, Notch is required for maintaining the self-renewal as well as the identity of type II NBs throughout larval development, but does not regulate the expression of Dpn in type II NBs.
To further explore the possibility that Dpn functions independently of Notch in type II NBs, we also performed the following two genetic interaction tests. First, we examined whether loss of Dpn inhibits the over-proliferation of type II NBs caused by over-expression of activated Notch. Over-expression of activated Notch, the intracellular domain of Notch (NIC), or loss of Numb leads to the overproliferation of both type I and type II NBs [4], [28], [29]. We found that over-expressing NIC using insc-Gal4 ase-Gal80 resulted in similar over-proliferation of type II NBs in wild type and dpn mutant larval brains (Fig. 8A–B'). Second, we examined whether lowering Notch expression enhances the loss of type II NBs in dpn7 heterozygous larval brains. dpn7 heterozygous mutant larvae contained a slightly reduced number of type II NBs (about 6 per brain lobe) (Fig. 8C), thus providing a sensitized genetic background. However, we found no further reduction of the number of type II NBs in Notch dpn7 trans-heterozygous mutant brains. These genetic data further suggest that Dpn and Notch function in separable pathways to regulate type II NB self-renewal and identity.
Dpn was initially identified as a pan-neural protein about two decades ago [15] and has been widely used as a NB marker. However, the function of Dpn in NBs has been elusive. In this study, we provided evidence that Dpn plays an important role in maintaining NB self-renewal. In type II NBs, in addition to maintaining the self-renewal, Dpn is also required to suppress Ase expression when these NB exit quiescence. Furthermore, we demonstrate that Notch and Dpn may function independently in larval NBs. While both Dpn and Notch are required for maintaining the identity and self-renewal of type II NBs, knockdown of Notch does not affect the expression of Dpn in type II NBs.
In a developing nervous system, NSCs must be maintained when they divide in order to generate the complete array of neurons and glia that form a functional neuronal circuit. Current studies are focused on determining how NSC self-renewal is maintained, as well as mechanisms governing NSC terminal differentiation (reviewed in [30]). Our findings that dpn mutant MB NBs as well as other type I NBs are premature progressive lost demonstrate that Dpn functions cell-autonomously to maintain the self-renewal of larval NBs. Interestingly, in dpn mutant larvae, the premature loss of type I NBs mainly occurred within 48 hours ALH (with the exception of the MB NB). Zacharioudaki et al. [20] reported recently that Dpn and E(spl) proteins function redundantly to maintain NB self-renewal but have different temporal expression patterns. Dpn expression in NBs is activated at the newly hatched larval stage, whereas E(spl)mγ expression becomes obvious only when NBs start to divide at the 2nd instar larval stage [20]. The difference in temporal expression patterns between Dpn and E(spl) proteins probably explains why loss of type I NBs occurred mainly within 48 hours ALH in dpn mutants. Interestingly, despite the redundant function of Dpn and E(spl) proteins in maintaining NB self-renewal, loss of Dpn alone resulted in the premature loss of MB NBs at late larval/early pupal stages, indicating that E(spl) proteins may not be involved in maintaining MB NBs at late larval/early pupal stages. Our findings as well as those of Zachariousdaki et al. [20] suggest that Dpn is required for maintaining NB self-renewal rather than NB formation or specification as was proposed by San-Juan and Baonza [21]. It is likely that differences in identifying and quantifying NBs at different developmental stages accounts for this discrepancy.
The role of Dpn in NB self-renewal is also supported by the observation that ectopic/over-expression of Dpn promoted non-dividing immature INPs and terminally dividing GMCs to enter self-renewing divisions, and prolonged the self-renewal of both types of NBs. These ectopic self-renewing GMCs and immature INPs, which normally do not express Dpn, may de-differentiate to acquire a NB-like fate and contribute to the increased number of NBs, similar to what has been observed in brat, numband klumpfuss mutant type II NB lineages [4], [31]. However, type II NB lineages show more severe over-proliferation phenotypes than type I NB lineages in response to ectopic/over-expression of Dpn. The difference in degree of over-proliferation between the type I and type II NB lineages is likely related to intrinsic differences between the type I and type II NB daughters, rather than a difference in how Dpn itself is acting. Type I NBs produce GMCs that express genes such as pros and ase that limit proliferation [12], counteracting the pro-self-renewal function of Dpn. In contrast, type II NBs and immature INPs express the ETS family protein Pointed [16] but do not express Ase or Pros, making them particularly susceptible to the ectopic expression of genes, such as dpn, that promote self-renewal. We propose that the significantly enhanced proliferation of type II NB progeny in response to ectopic/increased Dpn expression is most likely due to a disparity in the inherent self-renewal potential of the type I and type II NB daughters.
The function of Dpn in maintaining NB self-renewal is consistent with mammalian Hes family proteins' function in maintaining NSCs. In the developing mammalian nervous system, the loss of Hes1, Hes3, and Hes5 leads to accelerated neurogenesis and premature depletion of neuroepithelial cells and radial glial cells, whereas forced expression of Hes proteins maintains NSCs [32], [33], [34], [35], [36], [37].
While Dpn is expressed in both type I and type II NBs, our data showed that the loss of Dpn not only resulted in the premature loss of type II NBs at early larval stages, but also led to the ectopic expression of type I NB markers Ase and Pros in type II NBs when they exited quiescence, making type II NBs appear as type I-like NBs. This indicates Dpn has two roles in type II NBs: Dpn maintains NB self-renewal just as it does in type I NBs, and Dpn is also required to maintain type II NB identity. Moreover, it appears that Dpn's role in maintaining type II NB identity is temporally restricted. Results from our study as well as others [21] showed that dpn mutant embryonic brains contained a comparable number of Dpn+ Ase- NBs as wild type embryonic brains. In dpn mutant clones, our results showed that type II NBs did not ectopically express Ase even 4 days after clone induction when Dpn is no longer detectable. Therefore, it seems that Dpn's function to suppress Ase expression is limited to a narrow temporal window during the reactivation of type II NBs at the 1st instar larval stage. How might Dpn act to maintain type II NB identity? In mammals, Hes family proteins are well known for their roles in antagonizing the expression and/or activity of proneural genes (Ase is a member of the achaete-scute family of proneural genes) [38]. Negative interactions between dpn and the achaete-scute complex (AS-C) genes occur during Drosophila sex determination as well as neurogenesis [15], [39]. One potential model could be that a proneural protein(s) might be expressed in quiescent type II NBs and that Dpn is required to antagonize its expression and/or activity in order to promote type II NB fate when NBs exit quiescence. Since Dpn is expressed in both type I and type II NBs, we postulate that its role in maintaining type II NB fate is associated with the differential expression and/or activity of another, currently unidentified gene.
Our work suggests that the premature loss of dpn mutant type I NBs could be mediated by Pros. This is supported by our findings that nuclear Pros precociously accumulates in dpn mutant type I NBs and that dpn mutant type I NBs are maintained even in adult brains in the absence of Pros. It has been shown that over-expressing Pros in embryonic and larval NBs is sufficient to induce ectopic nuclear Pros localization and terminal division [12], [40]. Therefore, one possibility is that Dpn negatively regulates pros expression. In the absence of Dpn, Pros expression increases, leading to the nuclear accumulation of Pros and thus premature terminal division. In type I NBs, dynamic cortical and cytoplasmic localization of Pros makes it difficult to compare the levels of Pros in wild type and dpn mutant type I NBs by immunostaining. However, ectopic Pros expression in dpn mutant type II NBs, which normally do not have Pros, provide evidence that Dpn negatively regulates Pros expression, either directly or indirectly. The existence of putative Dpn binding sites in the pros promoter suggests that Dpn could directly regulate pros expression [41]. Alternatively, Dpn could indirectly regulate pros by inhibiting the expression and/or activity of proteins, such as Ase, that promote pros expression. In support of this notion, it has been shown that mammalian Hes proteins can inhibit the expression of proneural proteins such as Mash1 in the developing cortex [42], whereas forced expression of the proneural protein Mash1 in neuroepithelial cells is sufficient to promote the expression of Prox1, the mammalian homolog of Pros that plays an anti-proliferative and pro-differentiation role in the developing mammalian hippocampus and retina [43], [44], [45], [46].
Unlike the majority of mammalian Hes proteins or other members of the fly Hes family, which typically act downstream of Notch [34], [38], [47], results from this study as well as Zacharioudaki et al. [20] do not support a model in which Dpn functions as a direct target of Notch signaling in larval NBs as was proposed by San Juan and Baonza [21]. First, although our studies, as well as the work from other investigators [48], showed that the knockdown of Notch or disruption of Notch signaling led to premature loss of type II NBs and ectopic expression of Ase in type II NBs as was observed in dpn mutant larval brains, knockdown of Notch did not affect the expression of Dpn in type II NBs, which is consistent with previous findings [20]. Second, our data and those of Zacharioudaki et al. [20] showed that removing Dpn did not abolish the over-proliferation of type II or type I NBs caused by over-expression of activated Notch. Nor did reducing Notch expression exacerbate the loss of type II NBs in dpn7 heterozygous animals. These genetic interaction data suggest that Dpn does not function downstream of Notch signaling. Thus, Dpn may be similar to the mammalian Hes2 and Hes3, which are not transcriptionally regulated by Notch [49].Notch and Dpn likely employ distinct mechanisms to maintain the self-renewal and suppress Ase expression in type II NBs. Zacharioudaki et al. [20] showed that some E(Spl) proteins, particularly E(spl)mγ and m8, depend on Notch signaling for their expression in larval NBs. However, loss of E(Spl) proteins does not result in ectopic expression of Ase in type II NBs. Therefore, Notch must function through molecules, which are yet to be identified, to regulate Ase expression in type II NBs.
Dpn protein perdures in dpn1 mutant clones. (A-B') Dorsal views of a wild type (A) and dpn1 mutant (B) 3rd instar larval brain. Dpn is detected in wild type brains (A') but not in dpn1 mutant brains (B'). Note Ase-negative type II NBs (arrows in A) are absent in the dpn1 mutant brain (B). (C-F') dpn1 mutant type I (C-D') and type II (E-F') NBs clones at 2 days (C, C', E, E') or 4 days (D, D', F, F') after clone induction. Dpn protein is detected at reduced levels in both type I (C, C') and type II (E, E') NBs at 2 days after clone induction, but not at 4 days after clone induction (D, D', F, F'). Both type I (D, D')and type II (F, F') NBs remain present in dpn1 mutant clones at 4 days after clone induction and type II NBs remain Ase- (F, F').
Loss of type II NBs in dpn mutant 3rd instar larval brains. (A–A') mCD8-GFP driven by insc-Gal4 in combination with ase-Gal80 labels the type II NB lineages in a 3rd instar larval brain. Arrows indicate the Ase-negative type II NBs. (B–B') In dpn mutant 3rd instar larval brains, no NBs are labeled by mCD8-GFP driven by insc-Gal4 with ase-Gal80.
dpn mutant type II NBs ectopically express nuclear Pros at the 1st instar larval stage. (A–A") Wild type type I NBs (arrows) express nuclear Pros at 9–12 hours ALH. (B–B") Pros is not expressed in wild type type II NBs (arrowheads) at the same stage. (C–C") Ectopic nuclear Pros in a remaining dpn mutant type II NB (arrowhead) at 9–12 hours ALH. Type II NBs were labeled with mCD8-GFP expression (in green) driven by insc-Gal4 ase-Gal80. NBs are stained with either Dpn (A–A") or Mira (B–C").
Loss of Dpn does not affect the generation and specification of type II NBs in embryonic brains. A wild type (A–A") or a dpn mutant (B–B") brain lobe at embryonic stage 14/15 (10.5–13.5 hrs AEL) is stained with Ase in red and Dpn in green. Arrows point to Dpn+Ase- NBs. Wild type and dpn mutant brains contain similar numbers of Dpn+Ase- NBs.
dpn mutant MB NBs produce truncated lineages. (A–B) Diagrams showing that individual MB NBs sequentially generate three distinct types (γ,α'/β', and α/β) of MB neurons that target their axons to their corresponding lobes (B). (C–C'') An adult wild type MARCM MB clone (in green) generated at the newly hatched larval stage has all three different types of MB neurons. The brain was counterstained with Fas II in red to label the γ and α/β axon lobes. (D–F) Adult dpn7 mutant MB clones show various degrees of loss of late-born neurons. Among total 25 dpn7 mutant clones examined, 17 clones show the loss of late-born α/β neurons as indicated by axon reduction in the center of the α/β lobe shown in a single focal plane (D–D'), 3 clones show a complete loss of α/β neurons (E), and 5 clones have only γ 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 γ neurons contain much fewer MB neurons compared to wild type clones.
pros mutant NBs persist in dpn mutant adult brains. (A–B) prosv17 (A) and dpn prosv17 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.
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 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 (arrowheads). (D–D') A type I NB clone overexpressing Dpn has a single NB (arrow) but an increased number of GMCs (arrowheads). (E–E') A type I NB clone overexpressing Dpn contains multiple NBs (arrows) and increased number of GMCs.
Ectopic Dpn expression causes immature INPs to become mitotically active. (A–A') phospho-histone H3 (p-H3) is not detected in Ase-negative immature INPs (arrows) in wild type type II NB lineages. (B–B') Anti-p-H3 labels many immature INPs (arrows) when Dpn is ectopically expressed. Type II NB lineages are labeled by mCD8-GFP driven by insc-Gal4 combined with ase-Gal80. NBs are outlined by dashed circles.
Type II NBs are prematurely lost at the late 3rd instar larval stage. (A–A") Type II NB lineages in control brains are labeled by mCD8-GFP driven by GAL414–94. GAL414–94 also drives the expression of FLPase, which excises the stop codon and results in the expression of β-gal under the control of the constitutively active actin promoter. Through this approach, β-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. (B–B") In brains expressing Notch-RNAi, there is very little mCD8-GFP expression and there are no β-gal+ NBs, indicating Notch is necessary for maintaining NBs.
We thank Yuh Nung Jan and Lily Jan for their support of this work. We also thank Juergen Knoblich for insc-Gal4 flies, Harald Vaessin for UAS-dpn flies, as well as the Bloomington Stock Center and the Developmental Studies Hybridoma Bank for fly stocks and antibodies. We thank Peter Soba for comments on the manuscript and other members of the Jan lab for helpful discussions and suggestions.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's Canada Institute for Scientific and Technical Information in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


text:
Design fob a textile fabric
Abstract available in
Claims available in
Description (OCR text may contain errors)
Des. 117,131
Oct. l0, 1939.
A. TABIN TEXTILE FABRIC Filed Aug. 50, 1939 Wai/2635566:
zf/Q Patented Oct. 10, 1939 Des.
UNITED STATES PATENT OFFICE DESIGN FOR A TEXTILE FABRIC Adrian Tabin, Chicago, Ill. o Application August 30, 1939, Serial No. 86,946
Term of patent 3% years To all whom it may concern: y The figure is a plan View of a textile fabric Be it known that I, Adrian Tabin, a citizen of showing my design. the United States, residing in Chicago, in the I claim:
county of Cook and State of Illinois, have in- The ornamental design for a textile fabric, as Vented a new, original, and ornamental Design shown. for a Textile Fabric, of which the following is ADRIAN TABIN,
a specification, reference being had to the accompanying drawing, forming a part thereof.
Original Image

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Thai Recipes: Thai Red Curry A Favorite Chicken Curry
|Cooking oil
|3 Tablespoon (Canola oil, peanut oil, safflower oil any one of these)
|Chicken breast
|1 Pound, bite sized pieces
|1 Pound (black tiger prawns)
|Thai red curry paste
|3 Tablespoon
|Brown onion
|1 Medium, thinly sliced
|Bamboo shoots
|14 Ounce, thinly sliced
|Garlic cloves
|4 , chopped
|Coconut milk
|14 Ounce (unsweetened)
|Chicken stock
|1 Cup (16 tbs)
|Fish sauce
|2 Tablespoon
|Minced ginger
|2 Tablespoon
|Palm sugar/Brown sugar
|2 Tablespoon
|Lemongrass stalk
|2 Small
|Kaffir lime leaves/Bay leaves
|Red bell pepper
|2 , thinly sliced
|Mushroom slices
|2 Cup (32 tbs) (Shitake, straw, beech, shimeji, crimini any mushroom of your choice)
|Chopped basil
|1 Cup (16 tbs) (Thai basil)
|For serving
|1⁄2 , cut into quarters
|Basil leaves
|1 Tablespoon
|For garnish
|1⁄4 , julienned
|Sesame oil
|To Taste (Optional)
1. In a wok, heat oil and saute onion on medium high heat, add in ginger and garlic and stir for 5 minutes.
2. Put in Thai curry paste and little chicken stock, stir well to mix, and cook for 2 minutes.
3. Reduce the heat to medium low, pour in coconut milk and mix.
4. Add in remaining stock, and bring to a boil.
5. Lower the heat and allow to simmer, add in lemongrass, fish sauce, kaffir lime leaves and brown sugar and stir.
6. Raise the heat to medium high heat and bring to boil, add in the chicken and let it simmer for 10 minutes.
7. Put in the bamboo shoots, stir and add in mushroom leave it for 8 to 10 minutes.
8. Sprinkle in the Thai basil, stir for 2 minutes.
9. Lower the heat to low simmer and put in the prawns and cook for 2 minutes.
10. Serve hot on a bed of rice, garnished with basil leaves, courgettes and drizzle with some sesame oil (if using).
11. Serve with lime wedges on side, to squeeze on top.


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GM Holden Chairman and Managing Director Mike Devereux lays down something of an ultimatum, declaring Australia will have to decide next year whether or not it wants an automotive industry. Speaking at the local launch of the Colorado 7 SUV, Devereux tells media the future of local car manufacturing may hinge on next year's federal election. "My hope is the auto industry is not an election issue. It is a bipartisan issue. Either way, there needs to be another broad-based ...
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EMDR & Mindfulness:
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NOTE: Live, teleseminar participants MUST attend each class in full in order to receive the 6.5 EMDRIA credits. NO PARTIAL CREDIT WILL BE GIVEN!!
NOTE: Live, teleseminar participants MUST attend each class in full in order to receive the 6.5 EMDRIA credits. NO PARTIAL CREDIT WILL BE GIVEN!!
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Completion of an EMDRIA Approved Basic EMDR Training is prerequisite for registration. To receive credits for this program, participatns must attend each session in its entirety.
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Four group consultations are scheduled via teleconference, the last and final consultation will be set at the training. A call in number and PIN will be provided at the training to access the teleconference.
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This is the complete EMDR Basic Training approved by the EMDR International Association (EMDRIA), formerly known as Level I and Level II, including an additional 10 hours on consultation. Participants will learn the EMDR psychotherapy approach, the basic eight-phase EMDR protocol, the Adaptive Information Processing Model, and how to incorporate EMDR into your practice. This course will consist of didactic presentations, live demonstrations, supervised practicum, and case consultations. Participants must attend the full training and attend five 2-hour consultation groups to receive a certificate of completion in EMDR Basic Training. Five 2-hour consultation groups will be provided by the trainer, included in the cost of the training. If other dates or times are needed to make up for the consultation hours, attendees can either schedule separate consultation with the trainer (or any EMDRIA approved Consultant with prior approval of the trainer) at the attendees cost. Approval of partial programs will not be granted.
Required reading: Eye Movement Desensitization and Reprocessing: Basic principles, protocols, and procedures, second edition, by Francine Shapiro, PhD, EMDR originator.
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Small Bright Teal Aluminum Kiddush Cup with Matching Silver Stripe
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This small bright teal colored aluminum Kiddush Cup features a single silver stripe near the bottom edge and has a modern shape with slightly curved top edges. Read more
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Product Description
This handsome Kiddush Cup is made from anodized aluminum and features a simple shape. The cup has a flat bottom, cylindrical sides and a top edge with a slightly rounded lip. The bottom of the cup is adorned with a matching silver stripe. This cup can be used to drink Kiddush wine or any other drink.
This item is an ideal holiday gift idea!
General Information
Material Aluminium
Dimensions 4x7cm
Estimated processing time:
3-5 Business Days.Learn More
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4th of July Crafts and Recipes
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Independence Feast Apron
By: DecoArt
We are adding the craft to your Craft Projects.
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Click here to view your Craft Projects.
This 4th of July craft project creates a colorful apron celebrating a "Feast for the Fourth." Wear during Independence Day cookouts and other celebrations for a bit of festivity.
DecoArt Products Required
SoSoft Fabric Acrylics
- DSS1 - White
- DSS2 - Antique Gold
- DSS23 - Dark Chocolate
- DSS24 - Lamp Black
- DSS35 - Soft Peach
SoSoft Metallics
- DSM11 - Bright Blue Metallic
Americana Acrylics
- DA128 - Deep Burgundy
Dazzling Metallics
- DA117 - White Pearl
Royal Metallics
- DM04 - Brilliant Silver
Americana Spray Sealers
- DAS13 - Americana Matte Spray
Fabric Painting Medium
- DAS10 - Fabric Painting Medium
- Brushes - by Loew-Cornell - Fabric Brush Series; FAB Flats - 4F & 8F; FAB Round - 2
- Brushes - La Corneille Series - Series 7450, 1/4" & 3/8" Angle; Series JS 1 Liner
- Supplies - Paper towels, palette or plastic plate, water container, old toothbrush or speckling brush 808866-8, nine 2" wooden stars, 1/8" & 1/4" small star stencil, powder blush for cheeks, silver thread, Loew-Cornell double-ended stylus, palette knife No. J-O (1-1/4"), belt sander, drill, transfer paper
- Denim Apron of Your Choice
- Pattern
1. Round edges of stars with 1" belt sander.
2. Drill two holes in center of stars to create buttons.
NOTE: When using paints that are not So-Soft, mix Fabric Painting Medium with paint, following instructions on bottle.
1. Transfer the design onto the apron using white transfer paper.
2. Basecoat all design areas with White and 8F brush. (Refer to illustrations.)
1. Basecoat dress, shoes and red stripes with 8F brush loaded with Deep Burgundy.
2. Basecoat white stripes with 2 round brush loaded with Buttermilk.
3. Use 4F brush to basecoat blue stripe on bottom of dress with Williamsburg Blue.
4. Use 1/4" stencil to stencil stars on blue stripe with Buttermilk.
5. With 3/8" angle brush, shade dress, shoes and red stripes with Black, highlight upper edges of sleeves with Buttermilk, and shade white stripes with Black and blue stripe with Bright Blue.
1. With 8F brush, basecoat pinafore with Buttermilk.
2. Use 1/8" star stencil to stencil stars on pinafore.
3. Shade around edges and waistline with Black using 3/8" angle brush.
4. Shade around edge of stars with Bright Blue using 1/4" angle brush.
1. With 4F brush, basecoat face, hands and feet with Flesh Tan, and hair with Dark Chocolate.
2. Use 1/4" angle brush to shade face, hands and feet with Dark Chocolate.
3. Using 1 liner brush, add highlights to hair with Antique Gold, Black and White.
4. With 4F brush, shade hair and dip dot eyes with Black.
5. Powder blush cheeks with Q-Tip.
1. With 8F brush, basecoat with White.
2. Transfer lettering and paint with Bright Blue.
3. Shade around edge of banner with 3/8" angle brush loaded with Williamsburg Blue.
1. Use 4F brush to basecoat three stars with Bright Blue, three with Metallic Burgundy, and three with White Pearl.
2. Use 1 liner to line all stars and paint garland wire with Brilliant Silver.
1. Using toothbrush or speckling brush loaded with black, spatter entire painted design. (Avoid getting too many spatters on the face.)
2. Sew all star buttons into place with silver thread.
3. Cut six strands of thread 8" long for each of the stars; bundle strands together and tie them around under each button. Trim to desired length.
4. Spray all painted areas with matte spray.
For the latest book by RazzleBerries (Janine Oppelt and Debby Stenberg), contact them at 21194 Turner Ave., Hilmar, CA 95824; (209) 634-5434
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text:
Edith Joann Clawson was born on November 30, 1959 in Monroe, Michigan. She was born from the union of the late P.J. Clawson and the late Alda (Edwards) Clawson. Jody graduated from Monroe High School in June of 1977. She married the love of her life, James Paul on April 21, 1978 at Grape Missionary Baptist Church. Together they raised three daughters.
Jody attended LaSalle Missionary Baptist Church and has been a dedicated member there for over thirty-five years. There she taught Sunday School and Vacation Bible School each summer. Faith was very important to Jody and she received much joy sharing her faith with others.
As Jody stayed home to raise her children, she blessed others by providing babysitting services at her residence. After the children had grown, she helped support her family as a cashier at Walmart for more than ten years.
Jody was a very kind and loving person, always having nice words to say about everyone she met. Jody relished the time that she shared with her family, and loved to spend time with her grandchildren.
Edith "Jody" Joann Paul, age 54 of Monroe, passed away on Sunday, May 4, 2014 at home under the care of Heartland Home Hospice. In addition to her parents, she was preceded in death by a sister: Brenda Pridemore.
To cherish her memory, Jody leaves her beloved husband of thirty-six years: James Paul of Monroe; three daughters: Misty (Ed Henry) Remnant, Jennifer (Scott) Brown of Monroe and Amanda Paul of Monroe; two sisters: Sondra (Junior) Chumley and Peggy (Timothy) Tyniw; six brothers: Roger (Chris) Clawson, Phillip (Penny) Clawson, Larry Clawson, Randy (Kimberly) Clawson, Jeffrey Scott (Tina) Clawson and Lee (Sandy) Clawson; nine grandchildren: Grace Brown, Avery Brown, Katelyn Remnant, Kiley Remnant, Edward "E.J." Russell Henry Jr., Hailey Henry, Feliscity Henry, Mary Henry and Cody Henry.
Friends may gather from 3:00pm until 8:00pm on Wednesday, May 7, 2014 at Merkle Funeral Service, 14567 South Dixie Highway (734) 241-7070, formerly Bennett Funeral Services. A Celebration of Her Life will take place at 11:00am on Thursday, May 8, 2014 also at Merkle's with Reverend Joseph Baird of LaSalle Missionary Baptist Church officiating. Cremation will follow through Southern Michigan Services in Livonia, Michigan.
Online guests may leave words of inspiration and comfort, share a photo, or make a donation by visiting www.merklefs.com; Online Tributes page.


text:
Disregard BC-US--People-Celine Dion. A story slugged BC-US--Celine Dion-Shows Canceled moved with a Las Vegas dateline.
The AP
Friends trying to hand off rice cooker cause scare at casino
LINCOLN, R.I. (AP) — Police say two friends trying to hand off a rice cooker at a Rhode Island casino caused a brief security scare. Full Story
(Kevin Wolf/AP)
Billy Joel weds girlfriend during Fourth of July party
NEW YORK (AP) — Billy Joel married girlfriend Alexis Roderick in a surprise ceremony at the couple's annual Fourth of July party. Full Story
(Photo by Darren Carroll/Getty Images)
Did Tiger Woods cheat with Amanda Dufner?
Lindsey Vonn reportedly caught golfer with his competitor's ex-wife, which is likely why they aren't dating anymore.


text:
Analyst Research
|Report Title
PME African Infrastructure Opportunities PLC: Business description, financial summary, 3yr and interim financials, key statistics/ratios and historical ratio analysis.
Provider: Reuters Investment Profile
Provider: Wright Reports
NYSE and AMEX quotes delayed by at least 20 minutes. NASDAQ delayed by at least 15 minutes. For a complete list of exchanges and delays, please click here.
PME African Infrastructure Opportunities Plc announces results of current tender offer
PME African Infrastructure Opportunities Plc:Says the current tender offer for the purchase of up to 26,639,797 ordinary shares (26 percent of the ordinary shares outstanding) at $0.28 per ordinary share (the Current Tender Offer) closed at 1.00 p.m. on Dec. 5.Says a total of 25,706,863 ordinary shares, comprising 25.09 percent of the ordinary shares outstanding, were tendered.Says it is expected that both the CREST settlement of the Current Tender Offer and the despatch of cheques in respect of certificated holdings, will take place on Dec. 11.Says as set out in the Current Tender Offer document, the 25,706,863 ordinary shares validly tendered will be cancelled.Says therefore, with effect from Dec. 11 the total number of voting rights in the company will be 76,753,897.Says the voting rights on all these shares are identical.
Latest Developments forPME African Infrastructure Opportunities PLC
Latest Key Developments inInvestment
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text:
| BryceShiver
Reviews: 1
|http://www.dvdbooth.com/Western/THE_BIG_VALLEY_DVD_COLLECTION/ – great supply of DVDs, from the fine quality and playability of the DVDs, it's absolutely perfect! I'm definitely glad to own a copy of this show, which is really the BEST! This is one of the best TV shows ever made. Need I say more?
Review posted on Thursday, May 30th 2013 at 2:42 am


text:
Cardamom (disambiguation)
From Wikipedia, the free encyclopedia
Cardamom may refer to:
- Cardamom (or cardamon), species within two genera of the ginger family Zingiberaceae, namely Elettaria and Amomum
- Black cardamom (or brown cardamom), a plant in the family Zingiberaceae having pods used as a spice
- Green cardamom, a plant in the family Zingiberaceae having pods used as a spice
- Dwarf cardamom, Alpinia nutans, not a spice.
- Cardamom Khmer (or Western Khmer), a Khmer language dialect spoken by some Khmer people
- Cardamom Hills, located in south-central Kerala, India
- Cardamom Mountains (or Krâvanh Mountains), a mountain range in the south-west area of Cambodia, near the border with Thailand
- Cardamom Town (theme park) in Norway
|This disambiguation page lists articles associated with the title Cardamom.
If an internal link led you here, you may wish to change the link to point directly to the intended article.


text:
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Bothered by a barking dog? Have an electric, gas, sewer, or water issue? Need information about city services? City of Alexandria residents and visitors can use Alexandria Connects to notify the City and receive status updates as the issues are resolved. Alexandria Connects is a free application that allows you to report community issues, pinpoint the issue location with GPS, and attach photos right from your android device
Tags: alexandria discovering
from 8 reviews


text:
Can A.
Recording, Mix & Mastering Engineer, Sound Designer, Composer/Arranger
I am a music Producer, Recording, Mix & Mastering Engineer, DJ, sound designer, and composer with over 5 years of professional experience doing work for Pop, Jazz, Fusion, Rock, Metal, Urban (HipHop), R&B, Electro, Dance music genres, long and short film scoring, drum and synth programming, sound design for films and games and audio restoration. I own a studio called ''iCan Studios'' where I use it for professional works. I have SSL AWS900+ mixing desk (legendary SSL G series EQ), Pro Tools HD3 …more
Work History & Feedback
Turkish singer / musician (children's songs)"We are extremely satisfied with the work Can has done for us. He is not only creative and hardworking but also an excellent communicator. We recommend him to everyone."
Fixed Price $400
Video Requires Audio FX Only"Very good audio from can he was very good"
Fixed Price $50
Tests Taken
|Percentile Score
U.S. English Basic Skills Test


text:
Gran, You've Got Mail!
By Jo Hoestlandt; Aurélie Abolivier (Illustrator); Y. Maudet (Translator)
(Delacorte Books for Young Readers, Hardcover, 9780385735650, 128pp.)
Publication Date: September 9, 2008
List Price: $14.99*
* Individual store prices may vary.
Shop Local
Enter your zip code below to find indies closest to you.
ANNABELLE'S FATHER WANTS her to master her computer keyboard. Annabelle thinks the chore will be more tolerable if she writes letters to someone. She chooses Gran, her great-grandmother. Of course, Gran is most definitely not online, so Annabelle prints and mails her letters off. At first, Gran takes her time answering—and she's in the habit of repeating herself—but soon the two are keeping up a steady correspondence. Letter by letter, a true, tender friendship evolves. Annabelle and Gran talk about everything: parents, movies, school, the past and the present. When Annabelle divulges a big secret—the reason she and her best friend are no longer speaking—Gran remembers a similar situation. And when Gran needs foot surgery, Annabelle begins to worry.
Jo Hoestlandt is a prolific author of books for children in her native France.
Aurélie Abolivier illustrates books for children in France.


text:
The 2015 IGLA Annual Meeting has been set for August 5, 2015 at the Eriksdalsbadet swimming center. While the exact time and meeting room will be announced with the formal agenda, the Annual Meeting will take place after the first day of swimming on competition on August 5th (sometime between 2 and 6pm).
Some items on the agenda will include site selection of the 2017 IGLA Championships, elections for the following IGLA Board positions: Co-Chair (2 seats); and Treasurer and Webmaster. All of these board positions are open seats.
Please contact the IGLA Co-Presidents Bobby Ryan and Damien Strogen.


text:
Whether you are thinking of BUYING, SELLING, or interested only in having questions answered by a local real estate professional, INTEGRITY REAL ESTATE SERVICES is committed to providing accurate information to you.
INTEGRITY REAL ESTATE SERVICES offers its services to you because we believe that information is vital to making the best decisions when considering a sale, purchase or remodel of your home.
As a locally-owned, independent real estate brokerage, we want to also be your resource for community information. Please check out our Community Info page to locate a variety of useful websites.
Feel free to contact any of our real estate professionals to assist you. Also, as the Owners of INTEGRITY, we want to hear from you when you have a suggestion, criticism or compliment regarding WORKING WITH INTEGRITY.
Betsy Guild & Jacek Przepiora
IDX information is provided exclusively for consumers' personal, non-commercial use and that it may not be used for any purpose other than to identify prospective properties consumers may be interested in purchasing.


text:
Ciara And Future Named Their Son Future
The "Body Party" singer gave birth Monday morning in Los Angeles.
1. Maternity fashion icon Ciara gave birth to her first child yesterday in L.A.
But what's the baby's name? "Look in the album credits…" a source close to Future said.
2. And it appears that the newborn's name has been hidden in the liner notes of his dad's recently released album since April.
3. According to Future's liner note thank yous, his new son's name is also Future.
"To my kids Jakobi, Londyn, Prince, baby Future, & my adopted son Jaiden I love you guys more than life I promise, I promise, I promise!!," he wrote.
During her pregnancy, Ciara said she and Future had already picked a name. "I went with what felt right in my soul and my gut. I didn't second guess it," she told Us in May.
A rep for Future declined to comment.
Ciara confirms: "FUTURE ZAHIR WILBURN 9lbs 10oz May 19 2014."
6. Congrats, Cici!
Thumbnail image: Larry Busacca / Getty
- The U.S. has formally requested that Switzerland extradite 7 top FIFA officials who were arrested in Zurich in May.
- The U.S. government is investigating possible unlawful coordination by some airlines to keep prices high ✈
- Leaders of the U.S. Episcopal Church, which appointed an out gay bishop in 2003, have voted to let clergy perform religious same-sex marriages.


text:
Tags » Syleena's Fashion Style
Today is the day – I am moving my blog to my new homepage!
I hope you like it :)
Look #418
I love shoes….here comes a new release by Glamistry – Ramonda! 47 more words
Look #417 - X-Mas special
Dead Dollz offers a X-Mas special: some gorgeous gowns/dresses are available in a special christmas collection. 108 more words
Look #416
L'accessoires started into the december round… 56 more words
Look #415
Room69 ends tomorrow!!! 36 more words


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You can now invite anyone to meet at your favorite Starbucks.
Limited-time feature
26 year old man | Royse City, TX, USA | Seeking women 21-27 within 75 miles
Willing to lie about how we met!
[In reference to headline] But only if it's completely outlandish and hilariously intricate. Don't worry, we can sort out the details later. ;) I guess this is the part where I'm supposed to talk seriously about myself... Wellll, I'm a Texas guy through and through-it's always bigger and better, right? Anyway, I've been a nurse in a pediatric hospital for about 2 years and love my job. I'm pretty easy going and laid back, but I have my priorities straight and know exactly what I want in life. And I'm not afraid to work hard to get it! I'm looking to take on a second job, in hopes of saving enough money to buy some land and a house within the next year. I am very family- and goal-oriented and would like to meet someone along the same lines. I love being active and outdoors-jogging, backpacking, and hiking are a few of my favorite things. Unfortunately, daylight and I are no longer well acquainted since I work nights, and you apparently look like a creeper roaming around neighborhoods in the dark by yourself. Who would have thunk it??? I also love to travel and in the last few years I've been fortunate enough to visit Yellowstone National Park; Amsterdam; Ireland; the Caribbean's (multiple times); and, most recently, Las Vegas. A medical mission is probably in the near future as well. I'm never opposed to meeting new people and making new friends. However, I have a dry sense of humor and can be quite sarcastic at times so please don't take it to heart. I like to keep busy so if I'm not working I'm usually out with family or friends. Now, as for what I'm looking for from YOU ... -Education beyond high school is highly valued, though not necessary -Conversational skills are a must: if you can't have one without checking FB 10 times, applications will be rejected (If you don't know what the aforementioned abbreviation stands for, happily read on.) -A sense of humor and touch of sass never hurt anyone, either -Strong-willed and confident (without being arrogant) individuals are a plus -I'm looking for a lady that is as comfortable in the country as she is in the city -Honesty and loyalty are of utmost importance -Materialism is not welcome. I'm all about having pretty things in life, but the content of the relationships in your life is much more important If you have any questions, comments, or concerns, feel free to message me...
Camping , Cooking , Dining out , Fishing/Hunting , Gardening/Landscaping , Movies/Videos , Museums and art , Music and concerts , Exploring new areas , Playing cards , Playing sports , Travel/Sightseeing
Baseball, Billiards / Pool, Football, Running, Walking / Hiking, Weights / Machines
I exercise 3-4 times per week.
Don't display my sign
I like Dogs
Middle of the Road
Anywhere I can escape the hustle and bustle of every day life
Texas Country, Cars, Foods of all kinds, Working with my hands, Anything outdoors
I'm typically spending time with my family or hanging out with friends. On occasion, I'll run off on my own and enjoy some peace and quite on the road.
Currently reading "A Song of Fire and Ice" series (Game of Thrones)
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What he is looking for
|6'1" (185cms)
|4'11" (149cms) to 5'11" (180cms)
|Body type:
|About average
|Slender, About average, Athletic and toned
|No preference
|Black, Light brown, Dark brown, Blonde, Dark blonde
|No Way
|No Way, Occasionally
|Social Drinker
|Social Drinker, Moderately
|Medical / Dental / Veterinary / Fitness
|No preference
|I'll tell you later
|No preference
|Never Married
|Never Married, Widow / Widower, Divorced
|Have kids:
|No preference
|Wants kids:
|Definitely, Someday
|White / Caucasian
|Asian, East Indian, Latino / Hispanic, Middle Eastern, Native American, White / Caucasian
|Christian / Other
|No preference
|Bachelors degree
|Some college, Associates degree, Bachelors degree, Graduate degree, PhD / Post Doctoral


text:
ITHACA – The Ithaca City Police Department has opened an investigation into how a 39-year-old man came to be found dead in the bathroom of a downtown Ithaca business.
According to police, officers received a report from a business in the 200 block of West Seneca St. around 2 p.m. Tuesday, Dec. 17, that an unidentified man was unconscious in the bathroom. On the scene, officers found the man to be without a pulse. Emergency workers on the scene began CPR before Bangs Ambulance transported the man to Cayuga Medical Center, where he was pronounced dead. Police have not released the individual's name, pending notification of his family. The cause of death has yet to be determined but does not appear the result of any intentional criminal act, according to police.
The individual is a white male, 39 years old with hazel eyes, brown hair and a thin build. When found, he was wearing blue jeans, a black vest and a red plaid shirt, police said.
Anyone with pertinent information that would aid the police's investigation is asked to call Dispatch (607-272-3245) or Administration (607-272-9973).


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Monthly Archives: February 2013
Belle brokers world peace
I stood up to address the warring parties with a solution as simple as it was obvious. "You must all learn that it's OK when others don't agree with you"
Posted in Uncategorized Tagged peace, war Leave a comment
A conference encounter with a successful project manager (Rousse)
What fun! I turned up to the conference to find that I was seated between my primary school friend HP, and MH from my history A level class. I had seen HP relatively recently, but had had no contact with … Continue reading
Posted in Uncategorized Tagged A level, conference, friend, Friends, history, primary, project manager, school Leave a comment
A missed appointment with Paul McCartney (Rousse)
I'd waited years to meet Sir Paul McCartney in person and now I'd blown it. I thought that I had time before my appointment with the ex-Beatle to pop down to the harbour and check for a consignment of chewing … Continue reading
Posted in Uncategorized Tagged appointment, Beatle, boat, chewing gum, consignment, ex-Beatle, ferry, harbour, hero, Ireland, land, late, miss, Paul McCartney, sail, Sir Paul McCartney, time Leave a comment
Muddy bush women prove excellent runners (Rousse)
I was doing well, hill training with LM and SS in the woods. On the way back up through the trees I bounded past the pair of them with hardly a need to draw breath. My athletic achievement was soon … Continue reading
Elton John's red and white singalong at Covent Garden (Rousse)
When the three of us emerged from the tube station I instantly recognised Covent Garden. This was nowhere near High Street Kensington, our planned destination. VAE was confident that she would persuade the station staff to let us back in … Continue reading
Posted in Uncategorized Tagged audience, auditorium, band, concert, Covent Garden, crowd, crowds, doorway, Elton John, hall, High Street Kensington, master, palm, palm tree, palm trees, palms, performance, piano, platform, pop, pushchair, red, sign, signs, sing, singalong, stop, superstar, ticket, tickets, toddler, train, tree, trees, truth, tube, wail, white Leave a comment
Marie Helvin shops for shoes (Rousse)
Marie Helvin browsed the display of shoes that we had just admired. My sister-in-law SMcC wasn't going to miss this golden opportunity to speak to the "face of the seventies". She turned to the former supermodel and struck up a … Continue reading
Posted in Uncategorized Tagged ankle, boot, boots, conversation, design, display, face of the seventies, grey, heel, Marie Helvin, model, pink, shoe, shoes, shop, shopping, sister-in-law, supermodel Leave a comment
Peep Show's Super Hans seeks new recruits to his "Moonys" cult (Rousse)
It was a huge journey from Prague overland, but I eventually made it back to the UK – even though I was minus my pale green fake fur coat, lost by a fat middle-aged woman at a ball in Austria. … Continue reading
Posted in Uncategorized Tagged Austria, autumn, Balham, ball, bed, beds, Bertie, Birmingham, bus, campus, car, centre, city, coat, concession, cotton, cult, dress, fake, fake fur, footwear, fur, gap, garden, gay, geoologist, ginger, green, High Street Kensington, Kensington, lesbian, lettering, mall, matt king, middle-aged, passenger, passengers, pay, Peep Show, Prague, recruit, recruits, red-haired, redhead, roof, Russell and Bromley, shoes, shop, shopping, station, summer, Super Hans, term, transit van, trip, tube, university, van, warehouse, woman, work Leave a comment