Brain tumor specifies intermediate progenitor cell identity by attenuating β-catenin/armadillo activity - PDF

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epress. Posted online 20 November Published by The Company of Biologists Ltd (2014) 141, 1-12 doi: /dev RESEARCH ARTICLE STEM CELLS AND REGENERATION Brain tumor specifies intermediate

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epress. Posted online 20 November Published by The Company of Biologists Ltd (2014) 141, 1-12 doi: /dev RESEARCH ARTICLE STEM CELLS AND REGENERATION Brain tumor specifies intermediate progenitor cell identity by attenuating β-catenin/armadillo activity Hideyuki Komori 1, Qi Xiao 2, Brooke M. McCartney 3 and Cheng-Yu Lee 1,2,4, * ABSTRACT During asymmetric stem cell division, both the daughter stem cell and the presumptive intermediate progenitor cell inherit cytoplasm from their parental stem cell. Thus, proper specification of intermediate progenitor cell identity requires an efficient mechanism to rapidly extinguish the activity of self-renewal factors, but the mechanisms remain unknown in most stem cell lineages. During asymmetric division of a type II neural stem cell (neuroblast) in the Drosophila larval brain, the Brain tumor (Brat) protein segregates unequally into the immature intermediate neural progenitor (INP), where it specifies INP identity by attenuating the function of the self-renewal factor Klumpfuss (Klu), but the mechanisms are not understood. Here, we report that Brat specifies INP identity through its N-terminal B-boxes via a novel mechanism that is independent of asymmetric protein segregation. Brat-mediated specification of INP identity is critically dependent on the function of the Wnt destruction complex, which attenuates the activity of β-catenin/armadillo (Arm) in immature INPs. Aberrantly increasing Arm activity in immature INPs further exacerbates the defects in the specification of INP identity and enhances the supernumerary neuroblast mutant phenotype in brat mutant brains. By contrast, reducing Arm activity in immature INPs suppresses supernumerary neuroblast formation in brat mutant brains. Finally, reducing Arm activity also strongly suppresses supernumerary neuroblasts induced by overexpression of klu. Thus, the Brat-dependent mechanism extinguishes the function of the selfrenewal factor Klu in the presumptive intermediate progenitor cell by attenuating Arm activity, balancing stem cell maintenance and progenitor cell specification. KEY WORDS: Apc2, Brain tumor, Drosophila, Wnt signaling, Intermediate progenitor cell, Neuroblast INTRODUCTION Tissue-specific stem cells often undergo asymmetric cell division to self-renew and to generate an intermediate progenitor cell, which possesses limited developmental potential and functions to generate differentiated cell types (Ming and Song, 2011; Pierfelice et al., 2011; Weng and Lee, 2011; Homem and Knoblich, 2012). Thus, an efficient mechanism to abrogate the activity of self-renewal factors in the presumptive intermediate progenitor cell is pivotal for the maintenance of stem cell homeostasis and the generation of the 1 Center for Stem Cell Biology, Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA. 2 Department of Cell and al Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA. 3 Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA. 4 Division of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109, USA. *Author for correspondence Received 22 May 2013; Accepted 26 September 2013 requisite number of differentiated progeny. Failure to attenuate the function of self-renewal factors may contribute to the creation of tumor-initiating cells (Krivtsov et al., 2006; Wei et al., 2008; Liu et al., 2011; Haenfler et al., 2012; Xiao et al., 2012; Schwitalla et al., 2013). Thus, understanding the mechanisms that extinguish the activity of self-renewal factors in the presumptive intermediate progenitor cell is likely to provide novel insight into both normal development and tumor initiation. The type II neuroblast lineage in the fly larval brain provides an excellent model for investigating the mechanisms that regulate the proper specification of intermediate progenitor cell identity in vivo (Bayraktar et al., 2010; Weng et al., 2010; Song and Lu, 2011; Haenfler et al., 2012; Xiao et al., 2012). A type II neuroblast can be unambiguously identified by the presence of Deadpan (Dpn) and the absence of Asense (Ase) expression (Dpn + Ase ) (Bowman et al., 2008). Type II neuroblasts undergo repeated asymmetric cell division to self-renew and to generate early stage immature intermediate neural progenitors (INPs) (Dpn Ase ) that mature into late stage immature INPs (Dpn Ase + ) (Bello et al., 2008; Boone and Doe, 2008; Bowman et al., 2008; Bayraktar et al., 2010; Xiao et al., 2012). These late stage immature INPs acquire the functional identity of an INP (Dpn + Ase + ) and subsequently undergo limited rounds of asymmetric division to generate differentiated progeny. Thus, understanding the mechanisms that specify INP identity in immature INPs will provide insight into the proper specification of intermediate progenitor cell identity. TRIM32 and TRIM3, which are vertebrate orthologs of Drosophila Brain tumor (Brat), have been shown to play important roles in regulating neural stem cells during normal brain development and brain tumor formation (Boulay et al., 2009; Schwamborn et al., 2009). Brat contains two B-boxes and a coiledcoil domain in the N-terminus and an NHL domain in the C- terminus (Arama et al., 2000). The NHL domain is essential for Brat function in various developmental processes, including repression of mrna translation and regulation of microrna (Sonoda and Wharton, 2001; Neumüller et al., 2008; Harris et al., 2011). In mitotic neuroblasts, the NHL domain mediates the binding of Brat to the scaffolding protein Miranda (Mira), which partitions Brat exclusively into the progenitor cell (Betschinger et al., 2006; Lee et al., 2006a). Most of the previously isolated brat mutant alleles carry mutations in the NHL domain and exhibit defects in the maturation of immature INPs, leading to the formation of supernumerary type II neuroblasts (Arama et al., 2000; Bowman et al., 2008; Xiao et al., 2012). However, it is unclear whether the NHL domain contributes to the specification of INP identity directly or indirectly by promoting Brat protein accumulation in immature INPs. The activation of Wnt signaling plays crucial roles in both the regulation of stem cell self-renewal and the generation of differentiated cells (Merrill, 2012; Habib et al., 2013). In the absence of Wnt ligand, Wnt signaling is negatively regulated by the destruction complex, which includes the kinases GSK3β and CKI, 1 (2014) doi: /dev the scaffolding protein Axin, and the tumor suppressor Adenomatous polyposis coli (Apc) (Aoki and Taketo, 2007; MacDonald et al., 2009; Niehrs, 2012). This destruction complex phosphorylates β-catenin/armadillo (Arm) and targets it for proteosomal degradation. Binding of the Wnt ligand to the Frizzled receptor inactivates the destruction complex, leading to the accumulation and nuclear translocation of β-catenin/arm, where it complexes with Tcf/LEF family transcription factors and activates Wnt target gene expression. Despite its essential roles in regulating stem cell self-renewal and, ultimately, the generation of differentiated cells, little is known about the role of Wnt signaling in the specification of progenitor cell identity. Previous studies revealed that Drosophila Apc2 is cortically enriched asymmetrically in larval brain neuroblasts, such that a portion of the Apc2 is partitioned into the progenitor cell (McCartney et al., 1999). Furthermore, Apc2 becomes enriched in the cortex of the progenitor cell following neuroblast asymmetric division, suggesting that Wnt signaling activity might be negatively regulated in the progenitor cell (Akong et al., 2002). However, the functional significance of Wnt signaling in the progenitor cell has never been established. Here, we report that Brat specifies INP identity in immature INPs by downregulating Arm via a novel mechanism that is separable from the mechanism that regulates the asymmetric segregation of Brat. We identified that the B-boxes are dispensable for asymmetric partitioning of Brat into the presumptive immature INP but are necessary for Brat-dependent specification of INP identity. We further demonstrated that proper specification of INP identity by the Brat-mediated mechanism is critically dependent on Apc2, a key destruction complex component. Consistent with Apc2 negatively regulating Arm, reducing the function of the destruction complex or overexpressing constitutively active Arm enhances the supernumerary neuroblast phenotype in brat hypomorphic mutant brains. Furthermore, reducing arm function in immature INPs suppresses the supernumerary neuroblast phenotype in brat hypomorphic mutant brains, whereas increasing arm function in immature INPs strongly enhances the supernumerary neuroblast phenotype. These data strongly suggest that Brat specifies INP identity in immature INPs by attenuating the function of Arm through the destruction complex, thereby preventing the activation of Wnt target. Finally, removing arm function also strongly suppresses the supernumerary neuroblast phenotype induced by overexpression of the self-renewal factor Klumpfuss (Klu). Thus, Brat specifies INP identity in immature INPs by attenuating the function of the self-renewal factor Klu in part through extinguishing the transcriptional activity of Arm. RESULTS The B-boxes of Brat are dispensable for asymmetric protein segregation Since Brat unequally partitions into the immature INP following the asymmetric division of neuroblasts, the domains required for the asymmetric segregation of Brat are also likely to function to promote the specification of INP identity. We identified the domains required for asymmetric segregation of Brat in mitotic neuroblasts in brat null mutant brains by overexpressing a series of UAS-brat transgenes inserted into an identical docking site in the fly genome (Fig. 1A). Brat always colocalized with Mira in the basal cortex of telophase neuroblasts and co-segregated with Mira exclusively into Fig. 1. The coiled-coil domain and the NHL domain are both required to promote the asymmetric segregation of Brat in mitotic neuroblasts. (A) Schematics of Brat and the Brat deletion mutants used to identity the domain required to asymmetrically segregate Brat in mitotic neuroblasts in transgenic Drosophila and to map the Mira-binding domain in yeast two-hybrid analysis. Dashed lines indicate deletions. (B-C ) Brat and Brat B-boxes are cortically localized and segregate asymmetrically into the future immature INP in mitotic type II neuroblasts in brat null brains. (D-E ) Deletion of either the coiled-coil or the NHL domain (Brat C-coil or Brat NHL ) results in cytoplasmic localization and symmetric segregation into both daughter progeny. All Brat transgenic proteins are Myc tagged. The segregation pattern of the Brat transgenic proteins was determined based on the colocalization of the Myc epitope and Mira in telophase neuroblasts. Wor is a neuroblast-specific driver. Phh3, phosphohistone H3. (F) Yeast two-hybrid analysis showing that Brat and Brat B-boxes interact with both Mira and Mira , but Brat C-coil only interacts with Mira (G) Summary of the domains required for segregating Brat uniquely into the future immature INP during the asymmetric division of neuroblasts. Scale bar: 5 μm. 2 (2014) doi: /dev the future progenitor cell (Fig. 1B; 100%, N=12). Brat B-boxes also colocalized with Mira in telophase neuroblasts and co-segregated with Mira asymmetrically into the future progenitor cell (Fig. 1C; 60%, N=12). Thus, the B-boxes are dispensable for unequal partitioning of Brat during the asymmetric division of neuroblasts. By contrast, Brat NHL and Brat C-coil never colocalized with Mira in telophase neuroblasts and always segregated symmetrically into the cytoplasm of both daughter cells (Fig. 1D,E; 100%, N=12 per genotype). These results indicate that the NHL domain and the coiled-coil domain are essential for the asymmetric segregation of Brat in mitotic neuroblasts. The binding of Brat to the cargo-binding domain of Mira (amino acids ) is essential for its segregation into the future progenitor cell following the asymmetric division of neuroblasts (Betschinger et al., 2006; Lee et al., 2006a). Independently of our domain analyses, we identified that Mira and Mira (Fig. 1A) bind to both full-length Brat and Brat B-boxes in a yeast twohybrid screen using a brat mutant brain cdna library (H.K. and C.- Y.L., unpublished). We confirmed the interaction between fulllength Brat or Brat ΔB-boxes with both the Mira and Mira fragments in a one-to-one yeast two-hybrid interaction assay (Fig. 1F). These results indicate that the B-boxes are dispensable for the binding of Brat to Mira, consistent with Brat B-boxes colocalizing and co-segregating with Mira into the immature INP during the asymmetric division of neuroblasts (Fig. 1C). We next tested whether the coiled-coil domain of Brat mediates direct interaction with Mira or Mira Interestingly, Brat C-coil only interacts with Mira but not with the Mira fragment, indicating that the coiled-coil domain of Brat mediates the binding to amino acids of Mira (Fig. 1F). These data also suggest that the NHL domain most likely interacts with amino acids of Mira and are consistent with our previous study showing that glycine 774 and tyrosine 829 in the NHL domain of Brat mediate direct interaction with the cargo-binding domain of Mira (Lee et al., 2006a). We were unable to directly test the interaction between Brat NHL and Mira due to excessive auto-activation. Taken together, we propose that the coiled-coil domain and the NHL domain function cooperatively to target Brat into the future progenitor cell during the asymmetric division of type II neuroblasts. The B-boxes of Brat are uniquely required for the specification of INP identity We next tested whether the coiled-coil domain or the NHL domain is required for Brat-dependent specification of INP identity by overexpressing the UAS-brat transgene driven by a neuroblastspecific Wor-Gal4 driver in brat null brains (Fig. 1A, Fig. 2A). We previously showed that Ase immature INPs rapidly revert into supernumerary type II neuroblasts at the expense of INP formation in brat null brains (Fig. 2B,C,I; supplementary material Fig. S1A) (Xiao et al., 2012). Overexpression of brat suppressed the supernumerary neuroblast phenotype and restored INP formation in brat null brains (Fig. 2D,I; supplementary material Fig. S1B,C; N=10). Thus, restoring wild-type brat function in type II neuroblasts is sufficient to rescue defects in the specification of INP identity in brat null brains. Surprisingly, overexpression of brat C-coil or brat NHL also suppressed the supernumerary neuroblast phenotype in brat null brains (Fig. 2E,F,I; supplementary material Fig. S1D,E; N=10 per genotype). These results indicate that neither the coiledcoil domain nor the NHL domain is essential for Brat-dependent specification of INP identity. By contrast, overexpression of brat Bboxes failed to suppress the supernumerary neuroblast phenotype in brat null brains (Fig. 2G,I; supplementary material Fig. S1F,G; N=10 per genotype). Thus, the B-boxes are indispensable for Bratdependent specification of INP identity. Because the Brat protein has two B-boxes, we tested whether they might function redundantly to mediate Brat-dependent specification of INP identity. Overexpression of either brat B-box 1 or brat B-box 2 efficiently suppressed the supernumerary neuroblast phenotype and restored INP formation in brat null brains (Fig. 2H,I; N=10; data not shown). These data indicate that the B-boxes indeed function redundantly to mediate Brat-dependent specification of INP identity (Fig. 2J). Although all TRIM family proteins contain at least one B-box, the role of this domain in the function of these proteins is unknown. We tested whether the B-boxes might mediate the function of Brat in repressing the translation of hunchback mrna in the posterior of the preblastoderm embryo (Sonoda and Wharton, 2001). We overexpressed UAS-brat transgenes driven by nanos-gal4 in brat fs1/df mutant embryos. Consistent with previously published observations, overexpression of brat, but not brat NHL, rescued defects in abdominal segmentation in brat fs1/df embryos (supplementary material Fig. S1H-J). Importantly, overexpression of brat B-boxes or brat C-coil also rescued defects in abdominal segmentation in brat fs1/df embryos (supplementary material Fig. S1K-M). Thus, the B-boxes and the coiled-coil domain are dispensable for the function of Brat in repressing mrna translation. The B-boxes, therefore, uniquely elicit the function of Brat in the specification of INP identity (Fig. 2J). The mechanism by which Brat specifies INP identity is sensitive to reduced Apc2 function Our data thus far indicate that Brat specifies INP identity via a novel mechanism that is independent of the domains required for asymmetric protein segregation (Figs 1, 2). To gain mechanistic insight into how Brat specifies INP identity, we screened for haploinsufficient loci that modify the supernumerary neuroblast phenotype in a sensitized brat mutant background (Xiao et al., 2012). Briefly, a wild-type larval brain lobe contained 8±0 type II neuroblasts (data not shown). Whereas a hypomorphic brat DG19310 homozygous mutant brain lobe possessed 12±3 type II neuroblasts (supplementary material Fig. S2A,B; N=10), a genetically null brat 11/K06028 mutant brain lobe contained hundreds of type II neuroblasts (supplementary material Fig. S2A,C; N=10). A brat DG19310/11 mutant brain lobe possessed 24.5±4.7 type II neuroblasts and provides a sensitized genetic background for identifying the genetic modifiers of brat (Fig. 3A; supplementary material Fig. S2A; N=10). Through this screen, we identified Apc2 as a genetic enhancer of the supernumerary neuroblast phenotype in brat DG19310/11 mutant brains. We confirmed that reducing Apc2 function consistently enhanced the supernumerary neuroblast phenotype in brat DG19310/11 brains using multiple independently generated Apc2 mutant alleles, including a null allele [Apc2 g10 (McCartney et al., 2006)] and two hypomorphic alleles [Apc2 N175K and Apc2 d40 (McCartney et al., 2001; Hamada and Bienz, 2002)] (Fig. 3B; supplementary material Fig. S2A,D,E; N=10 per genotype). Furthermore, overexpression of Apc2 rescued the enhancement of the supernumerary neuroblast phenotype in brat DG19310/11 mutant brains induced by the heterozygosity of Apc2 (Fig. 3C; N=10). Thus, reduced Apc2 function enhances the supernumerary neuroblast phenotype in brat DG19310/11 brains. Despite sharing extensive sequence homology and functional redundancy with Apc2 (Ahmed et al., 2002; Akong et al., 2002; Hamada and Bienz, 2002), reducing Apc1 (Apc FlyBase) did not enhance the supernumerary neuroblast phenotype in brat DG19310/11 mutant brains (supplementary material Fig. S2A,F; N=10). In addition, reducing 3 (2014) doi: /dev Fig. 2. The B-boxes of Brat function uniquely in the specification of INP identity. (A) Summary of the UAS-brat transgenes used to test for B-box redundancy in the rescue of supernumerary neuroblasts in brat null brains. (B) Summary of the cell types in the type II neuroblast lineage in the Drosophila larval brain. Dpn, Deadpan; Ase, Asense; Pros, Prospero; GMC, ganglion mother cell; INP, intermediate neural progenitor; imm INP, immature INP; neurob, neuroblast. (C-H ) Overexpression of full-length brat, brat C-coil, brat NHL or brat B-box 2 rescues the supernumerary neuroblast phenotype in brat null brains, but overexpression of brat B-boxes does not. The effects on the supernumerary neuroblast phenotype in brat mutant brains were determined based on total type II neuroblasts per brain lobe. The dotted line separates the brain from the optic lobe where both are visible in the optical section. White arrows, type II neuroblast
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