I Reads About Neuro Blast Is This a Money Scheme or for Real ?

Evolution. 2012 Aug 1; 139(15): 2670–2680.

klumpfuss distinguishes stalk cells from progenitor cells during asymmetric neuroblast division

Qi Xiao

¹Department of Jail cell and Developmental Biology, Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, U.s.

Hideyuki Komori

⁴Middle for Stem Cell Biology, Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, Usa

Cheng-Yu Lee

^oneSection of Prison cell and Developmental Biology, Life Sciences Found, University of Michigan, Ann Arbor, MI 48109, Usa

²Program in Cellular and Molecular Biology, Life Sciences Institute, Academy of Michigan, Ann Arbor, MI 48109, The states

³Division of Molecular Medicine and Genetics, Department of Internal Medicine, Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA

⁴Center for Stem Cell Biology, Life Sciences Constitute, University of Michigan, Ann Arbor, MI 48109, USA

Supplementary Materials

Supplementary Material

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Abstract

Asymmetric stem cell segmentation balances maintenance of the stalk cell pool and generation of various prison cell types by simultaneously assuasive ane daughter progeny to maintain a stalk cell fate and its sibling to acquire a progenitor cell identity. A progenitor cell possesses restricted developmental potential, and defects in the regulation of progenitor prison cell potential can direct impinge on the maintenance of homeostasis and contribute to tumor initiation. Despite their importance, the molecular mechanisms underlying the precise regulation of restricted developmental potential in progenitor cells remain largely unknown. We used the type II neural stem prison cell (neuroblast) lineage in Drosophila larval brain equally a genetic model system to investigate how an intermediate neural progenitor (INP) cell acquires restricted developmental potential. We identify the transcription gene Klumpfuss (Klu) equally distinguishing a type Two neuroblast from an INP in larval brains. klu functions to maintain the identity of type Ii neuroblasts, and klu mutant larval brains show progressive loss of type II neuroblasts due to premature differentiation. Consistently, Klu protein is detected in type Ii neuroblasts but is undetectable in immature INPs. Misexpression of klu triggers immature INPs to revert to blazon II neuroblasts. In larval brains lacking brain tumor function or exhibiting constitutively activated Notch signaling, removal of klu function prevents the reversion of immature INPs. These results led the states to suggest that multiple mechanisms converge to exert precise control of klu and distinguish a progenitor jail cell from its sibling stalk cell during disproportionate neuroblast sectionalisation.

Keywords: Disproportionate cell division, Brain tumor, Intermediate neural progenitor jail cell, Klumpfuss, Neuroblast, Notch, Drosophila

INTRODUCTION

Asymmetric stem cell divisions provide an efficient mechanism for maintaining a steady stem cell pool while generating progenitor cells that requite ascension to differentiated progeny within the tissue where the stem cells reside (Morrison and Kimble, 2006; Pontious et al., 2008; Kriegstein and Alvarez-Buylla, 2009; Knoblich, 2010; Weng and Lee, 2011). Progenitor cells possess restricted developmental potential and role to protect the genomic integrity of stem cells by minimizing their proliferation. Since both daughter cells inherit the cellular content from their parental stalk cell during asymmetric sectionalisation, proper specification of sibling cell identity requires precise control of stalk cell determinants. Failure to properly downregulate stalk prison cell determinants in presumptive progenitor cells might allow them to acquire stem cell-like functional properties, and can perturb tissue homeostasis and contribute to tumor formation (Krivtsov et al., 2006; Wei et al., 2008). Thus, mechanistic insight into how the sibling cells assume singled-out identities during asymmetric stem cell partitioning is likely to accelerate our knowledge in stem cell biology, developmental biology and tumor biology.

In fly larval brains, two classes of neuroblast lineage tin be unambiguously identified based on the expression of cell fate markers and the backdrop of their progeny (Chia et al., 2008; Doe, 2008; Egger et al., 2008; Knoblich, 2010; Weng and Lee, 2011). A type I neuroblast expresses Deadpan (Dpn) and Asense (Ase) and divides asymmetrically to self-renew and to generate a progenitor cell called a ganglion female parent cell (GMC). By contrast, a type Two neuroblast (Dpn⁺ Ase⁻) divides asymmetrically to self-renew and to generate an immature intermediate neural progenitor (INP) that lacks the expression of Dpn and Ase and undergoes maturation during which it acquires an INP identity (Bello et al., 2008; Boone and Doe, 2008; Bowman et al., 2008). Following maturation, an INP (Dpn⁺ Ase⁺) undergoes limited rounds of asymmetric division to regenerate and to produce GMCs. A key functional property that distinguishes these 2 neuroblast lineages rests on their dependence on Notch signaling for the maintenance of their identity (Bowman et al., 2008; Vocal and Lu, 2011; Weng et al., 2011). Although dispensable for the maintenance of a blazon I neuroblast, Notch signaling is crucial for the maintenance of type 2 neuroblasts (Haenfler et al., 2012).

In mitotic type II neuroblasts, polarization of the cell cortex allows the basal proteins, including Brain tumor (Brat) and Numb, to segregate into the cortex of the presumptive young INP and promote the formation of INPs (Bello et al., 2006; Betschinger et al., 2006; Lee et al., 2006a; Lee et al., 2006c; Wang et al., 2006; Bowman et al., 2008; Wirtz-Peitz et al., 2008; Prehoda, 2009). Whereas a wild-type type 2 neuroblast is surrounded by three to five immature INPs and twenty to 30 INPs, a brat or numb mutant type Ii neuroblast is always surrounded past supernumerary neuroblasts at the expense of INPs. Thus, previous studies take proposed that brat and numb function in immature INPs, where these proteins promote the specification of an INP identity. All the same, the mechanisms by which brat and numb trigger an immature INP to assume the identity of an INP remain unknown.

In this study, we show that precise regulation of klu function is pivotal for distinguishing the self-renewing neuroblast from its sibling progenitor cell during asymmetric neuroblast division. Klu is necessary for the maintenance of type I and II brain neuroblasts, as klu mutant larvae showed progressive loss of both types of neuroblast. Klu is detected in all neuroblasts merely is absent-minded from their firsthand daughter progenitor progeny. Misexpression of klu in young INPs led to the germination of supernumerary type II neuroblasts. Importantly, removal of klu function prevented the reversion of young INPs to type II neuroblasts triggered by the loss of brat function or constitutive activation of Notch signaling. Furthermore, overexpression of klu also exacerbated the reversion of GMCs to blazon I neuroblasts every bit triggered by the aberrant activation of Notch signaling. Together, nosotros conclude that precise control of klu part by multiple signaling mechanisms distinguishes a neuroblast from a progenitor cell during disproportionate division of wing larval brain neuroblasts.

MATERIALS AND METHODS

Fly strains

Mutant and transgenic flies used include brat¹⁵⁰ (Betschinger et al., 2006), numb² (Skeath and Doe, 1998), klu^R51 (Kaspar et al., 2008), erm-GAL4 (Iii) (Pfeiffer et al., 2008), wor-GAL4 (Lee et al., 2006b), UAS-klu-HA, UAS-klu^one-583-HA and UAS-klu ^Δzf1 -HA (Kaspar et al., 2008), UAS-Notch_intra (Chung and Struhl, 2001) and UAS-cMyc (Benassayag et al., 2005). erm-GAL4 (Ii) was generously provided by Dr G. Rubin (HHMI). The following stocks were obtained from the Bloomington Drosophila Stock Center: Oregon R, brat^DG19310, deviling^k06028 (Arama et al., 2000), brat^eleven (Arama et al., 2000), Notch^55e11 (Artavanis-Tsakonas et al., 1984), klu⁰⁹⁰³⁶, Df(H99) (White et al., 1994), UAS-mCD8-GFP, UAS-apoliner (Bardet et al., 2008), UAS-p35, UAS-GFP, FRT19A (Lee and Luo, 2001), FRT2A, hs-flp (Lee and Luo, 2001), Deed-FRT-Cease-FRT-GAL4 (Pignoni and Zipursky, 1997), tub-GAL80 (Lee and Luo, 2001) and tub-GAL80^ts (Bloomington Drosophila Stock Center). Transgenic fly lines UAS-deviling-myc, UAS-HA-klu, UAS-HA-klu^1-583, UAS-HA-klu ^Δzf1 and UAS-HA-klu ^Δzf4 were generated using the pUAST-attB vector for insertion into an identical docking site in the fly genome via ϕC31 integrase-mediated transgenesis (Bischof and Basler, 2008).

Immunofluorescent staining and antibodies

Larval brains were dissected in Schneider'due south medium (Sigma), stock-still in 4% formaldehyde for 23 minutes and washed twice for twenty minutes each in 1× PBS containing 0.3% Triton 10-100 (PBST). After washing, brains were incubated with primary antibodies in PBST for iii hours at room temperature. Antibodies used include rat anti-Dpn (i:1000; this study), rabbit anti-Ase (1:400) (Weng et al., 2010), guinea pig anti-Ase (1:l; this study), mouse anti-Prospero (MR1A, 1:100) (Lee et al., 2006a), guinea pig anti-CycE (i:m; T. Orr-Weaver, Massachusetts Constitute of Technology, MA, U.s.), mouse anti-Dlg (one:fifty; Developmental Studies Hybridoma Bank), craven anti-GFP (1:2000; Aves Labs), rabbit anti-Klu (i:200) (Yang et al., 1997), rat anti-Mira (1:100) (Lee et al., 2006a), guinea pig anti-Numb (1:grand; J. Skeath, Washington Academy, WA, USA), rabbit anti-aPKC (1:one thousand; Sigma), mouse anti-phosphohistone H3 (ane:2000; Upstate Biotechnology), rabbit anti-PntP1 (1:600; J. Skeath) and rabbit anti-RFP (1:100; Rockland). Secondary antibodies were from Molecular Probes and Jackson Labs. We used Rhodamine phalloidin (1:100; Invitrogen) to visualize cortical actin. The confocal images were acquired on a Leica SP5 scanning confocal microscope.

Clonal analyses

Lineage clones were induced following the previously published method (Lee and Luo, 2001; Weng et al., 2010).

RESULTS

klu functions to maintain the identity of larval brain neuroblasts

Deviling is required cell-autonomously for the germination of INPs in larval brains (Betschinger et al., 2006; Lee et al., 2006c; Bowman et al., 2008). Thus, agreement how brat regulates the maturation of immature INPs will provide crucial insight into the mechanisms that distinguish the fates of sibling cells post-obit the asymmetric segmentation of type 2 neuroblasts. We assessed the identity of cells in the GFP-marked mosaic clones derived from a unmarried wild-type or brat naught mutant type Two neuroblast using the onset of Ase expression as a marker for an intermediate stage during maturation (supplementary material Fig. S1A-B; run across Discussion for more details). Each wild-type clone always contained one neuroblast surrounded by 2 to three Ase⁻ immature INPs, two to iii Ase⁺ immature INPs, INPs and GMCs (supplementary material Fig. S1C-D″,H; northward=seven per stage). By dissimilarity, a similarly staged brat mutant clone consisted of mostly neuroblasts, with very few Ase⁻ immature INPs and never whatsoever Ase⁺ young INPs or INPs (supplementary cloth Fig. S1E-I; north=vii per stage). These results led u.s.a. to conclude that Brat functions during maturation to forbid an young INP from acquiring a neuroblast fate while promoting it to assume an INP identity.

To elucidate the mechanisms past which Brat regulates the maturation of young INPs, we screened for haploinsufficient loci in the fly genome that modify the supernumerary type 2 neuroblast phenotype in a sensitized brat^DG19310/11 mutant genetic background (H.K. and C.-Y.L., unpublished). We identified klu as a genetic suppressor of brat, as heterozygosity of the klu locus strongly suppressed the formation of supernumerary neuroblasts in the brat-sensitized genetic background (supplementary cloth Fig. S1J-L; n=xviii per genotype). Thus, we propose that Brat regulates the maturation of young INPs past antagonizing klu.

To test whether Brat functions to prevent an immature INP from reacquiring a neuroblast fate or past promoting information technology to assume an INP identity, nosotros beginning analyzed the expression of prison cell fate and cell proliferation markers in wild-type and klu mutant larval brains (Fig. 1A). In wild-type larvae, the full number of neuroblasts reached the plateau of well-nigh 100 per encephalon hemisphere 72 hours after larval hatching (ALH) and remained at 100 per lobe at 96 hours ALH (Fig. 1B-B″,F; n=10 brains per phase). In similarly staged klu mutant larvae, total neuroblasts plateaued at ~80 per brain hemisphere at 72 hours ALH and decreased to less than lx per lobe at 96 hours ALH (Fig. 1C-C″,F; northward=ten brains per stage). Importantly, brain neuroblasts in wild-type or klu mutant larvae displayed similar proliferation profiles as indicated by the expression of Cyclin Eastward (CycE) and EdU pulse-chase labeling (Fig. 1D,E; 100% of neuroblasts in the brain, n=ten; information not shown). These results strongly suggest that klu is required for the maintenance of brain neuroblasts.

Neuroblasts prematurely differentiate in klu mutant brains. (A) Summary of the prison cell fate mark expression pattern in type I and 2 neuroblast lineages in Drosophila larval brains. GMC, ganglion mother cell; INP, intermediate neural progenitor; imm INP, immature INP; neurob, neuroblast; Pros, Prospero. (B-F) klu mutant brains show progressive loss of neuroblasts. (B-Eastward) Brains were dissected from wild-blazon or klu^R51/09036 mutant larvae at 96 hours ALH and stained for the markers indicated. The white dotted line separates the central brain (left) from the optic lobe (right). Discs large (Dlg) marks the cell cortex. (F) Boilerplate type I and II neuroblasts per brain lobe in larvae of genotypes and stages indicated. Error bars indicate s.due east.m. (G-L) Neuroblasts show reduced cell diameter and are likely to prematurely differentiate in klu mutant brains. Larvae conveying GFP-marked klu ^+/+ or klu^−/− mosaic neuroblast clones (outlined by the yellow dotted line) were aged for 110 hours after clone induction and larval brains were stained for the markers indicated. (G-H‴) Type I neuroblast clones. (I-K‴) Blazon II neuroblast clones. (L) The frequency of klu ^+/+ or klu^−/− clones containing neuroblasts of the cell bore indicated. The post-obit are indicated: type I neuroblast (Dpn⁺ Ase⁺), greenish arrow; GMC (Dpn⁻ Ase⁺), dark-green arrowhead; type II neuroblast (Dpn⁺ Ase⁻), white pointer; Ase⁻ immature INP (Dpn⁻ Ase⁻), white arrowhead; Ase⁺ immature INP (Dpn⁻ Ase⁺), yellowish pointer; INP (Dpn⁺ Ase⁺), yellow arrowhead. Calibration bars: 20 μm in B-E; x μm in Grand-K‴.

Nosotros side by side tested whether klu functions cell-autonomously to maintain encephalon neuroblasts by inducing GFP-marked mosaic clones derived from a unmarried wild-type or klu mutant neuroblast. Although both wild-type and klu mutant type I neuroblast clones maintained a single neuroblast per clone, 36.seven% of the klu mutant clones contained neuroblasts of reduced prison cell diameter (≤10 μm) (Fig. 1G-H‴,Fifty; due north=xxx clones per genotype). Similarly, half of the klu mutant type II neuroblast clones as well contained neuroblasts of reduced cell diameter (≤10 μm) (Fig. 1I-J‴,L; n=8 clones per genotype). Reduction in neuroblast bore was previously shown to correlate with the onset of premature differentiation (Lee et al., 2006b; Song and Lu, 2011). Consistently, 12.five% of the klu mutant clones contained multiple INPs, GMCs and their progeny (Fig. 1K-L; due north=8 clones). Together, these results led us to conclude that klu functions to maintain the identity of neuroblasts in larval brains and to suggest that Brat is likely to prevent an immature INP from reacquiring a neuroblast fate by antagonizing Klu.

Defects in cell polarity or aberrant activation of cell death can atomic number 82 to premature neuroblast loss in larval brain (Lee et al., 2006b; Bello et al., 2007), and so we tested whether klu maintains neuroblast identity by regulating cell polarity or jail cell survival. To appraise whether klu is required for polarization of the neuroblast cortex, we examined the localization of singular Protein kinase C (aPKC), Miranda (Mira) and Numb (Albertson and Doe, 2003; Rolls et al., 2003; Lee et al., 2006a; Lee et al., 2006c) in telophase neuroblasts in klu mutant brains. We detected aPKC segregated exclusively into the cortex of the time to come neuroblast and Mira and Numb localized asymmetrically in the cortex of the time to come progenitor cell in klu mutant brains (supplementary cloth Fig. S2A,B). Thus, since mitotic klu mutant neuroblasts displayed asymmetric localization of the apical and basal proteins, information technology is unlikely that klu maintains the identity of neuroblasts by regulating polarization of the neuroblast cortex. To determine if klu is required for the maintenance of neuroblast survival, we examined whether blocking activation of apoptosis would prevent the premature loss of neuroblasts in klu mutant brains. We generated mosaic clones derived from a single blazon I or Two neuroblast lacking klu alone or klu and the Df(3R)H99 locus. The H99 locus contains three crucial activators of apoptosis in the wing genome (White et al., 1994; Grether et al., 1995; Chen et al., 1996; White et al., 1996). However, removal of the H99 locus did not significantly subtract the occurrence of neuroblasts of reduced prison cell bore (≤x μm) or revert the absence of type 2 neuroblasts in klu mutant clones (supplementary fabric Fig. S2C-D‴; n=14 per genotype). Furthermore, we failed to observe abnormal activation of caspases in klu mutant brains, and blocking caspase activeness did not prevent premature neuroblast loss in klu mutant brains (supplementary cloth Fig. S2E-I; n=15 per genotype). Thus, we conclude that Klu does non maintain the identity of neuroblasts by regulating cell polarity or cell survival.

Overexpression of klu induces massive expansion of type Ii neuroblasts

Phenotypic analyses of klu mutant brains led us to conclude that klu functions to maintain the identity of neuroblasts in larval brains, so nosotros hypothesized that klu should exist expressed in both type I and II neuroblasts. Nosotros first assessed the spatial expression blueprint of the klu-lacZ enhancer trap line in larval brains. We detected lacZ expression in both blazon I and II neuroblasts as well as in their firsthand progenitor progeny in larval encephalon (supplementary cloth Fig. S3; northward=ten). Since the half-life of the β-gal poly peptide might exist longer than that of endogenous Klu protein, we stained larval brains carrying GFP-marked lineage clones derived from a single wild-type blazon I or II neuroblast with an antibody specific for Klu protein. In the type I neuroblast lineage, Klu was detected in the neuroblast but undetectable in GMCs and their progeny (Fig. 2A-A″,C; north=9 clones). In the type Ii neuroblast lineage, Klu was nowadays in the neuroblast and INPs just absent from immature INPs and GMCs (Fig. 2B-C; n=5 clones). Thus, we conclude that Klu is expressed in both types of neuroblast merely is absent from their immediate progenitor progeny.

Overexpression of klu induces supernumerary type Two neuroblasts. (A-C) Klu is detected in neuroblasts but is undetectable in their immediate progenitor progeny. (A-B‴) Drosophila larvae conveying GFP-marked wild-type type I or 2 neuroblast lineage clones (outlined by the yellowish dotted line) were aged for 72 hours after clone consecration and larval brains were stained for the markers indicated. (C) Summary of the Klu expression pattern in type I and II neuroblast lineages. (D-E″) Overexpression of klu induces backlog type II neuroblasts. Larvae of the genotype indicated were raised at 31°C for 72 hours ALH and larval brains were stained for the markers indicated. The white dotted line separates the fundamental brain (left) from the optic lobe (right). (F-Thou‴) Overexpression of klu specifically induces supernumerary neuroblasts in blazon II neuroblast lineage clones. Larvae carrying GFP-marked blazon I or II lineage clones (outlined by the yellow dotted line) overexpressing klu were aged for 24 hours after clone induction and brains were stained for the markers indicated. Abbreviations and arrows/arrowheads every bit Fig. 1. Scale bars: x μm in A-B‴,F-G‴; 20 μm in D-E‴.

The spatial expression blueprint of Klu is consistent with its proposed function in the maintenance of neuroblast identity, and then we tested whether increased office of klu can trigger the formation of supernumerary neuroblasts. We first overexpressed a UAS-klu transgene under the control of a pan-neuroblast wor-GAL4 driver in larval brains. Unexpectedly, we observed massive expansion of type Two neuroblasts but did non notice any increase in blazon I neuroblasts (Fig. 2d-East″; north=7 per genotype). Similarly, lineage clones derived from a single type I neuroblast overexpressing klu driven past a constitutively agile Actin-GAL4 driver reproducibly contained one neuroblast per clone (Fig. 2F-F‴; 100%, due north=x clones). By contrast, type II neuroblast clones overexpressing klu contained mostly neuroblasts (Fig. 2G-Yard‴; 100%, n=10 clones). Together, these results indicate that increased function of klu specifically leads to the expansion of type II neuroblasts.

Misexpression of klu in young INPs leads to supernumerary type II neuroblasts

We next examined the cell type from which supernumerary neuroblasts arise in the type Two neuroblast clones overexpressing Klu. We tested whether type 2 neuroblasts overexpressing Klu undergo symmetric division in telophase to generate supernumerary neuroblasts past analyzing the localization of aPKC, Mira and Numb. We observed that aPKC segregates into the cortex of the future neuroblast and Mira and Numb partitioning into the cortex of the future immature INP (Fig. 3B,C; n=15 per genotype). This result strongly suggests that a type II neuroblast overexpressing klu divides asymmetrically to generate a neuroblast and an young INP. We reproducibly observed Ase⁻ immature INPs in all type II neuroblast clones overexpressing klu (Fig. 2G-Chiliad‴). Thus, it is unlikely that type Ii neuroblasts overexpressing Klu undergo symmetric division to generate supernumerary neuroblasts. We adjacent tested whether supernumerary neuroblasts arise from de-differentiation of INPs in type II neuroblast clones overexpressing Klu. The lineage clones derived from INPs overexpressing klu maintained a single INP per clone and contained GMCs and their progeny only never type 2 neuroblasts, indicating that overexpression of klu is non sufficient to trigger INPs to de-differentiate back into type II neuroblasts (Fig. 3A-A‴; 100%, northward=viii). Thus, it is unlikely that supernumerary neuroblasts in type II neuroblast clones overexpressing Klu originate from symmetric neuroblast division or de-differentiation of INPs.

Misexpression of klu triggers the reversion of immature INPs to type II neuroblasts. (A-A‴) Overexpression of klu is not sufficient to trigger de-differentiation of INPs. Drosophila larvae carrying GFP-marked INP lineage clones (outlined past the yellow dotted line) overexpressing klu were aged for 24 hours after clone induction and brains were stained for the markers indicated. (B,C) Telophase neuroblasts overexpressing klu bear witness asymmetric localization of upmost and basal proteins. Phh3, phosphorylated histone H3. (D-F) The action of erm-GAL4 is first detected in immature INPs. (D-E‴) Larvae expressing GFP driven past erm-GAL4 (2) or erm-GAL4 (Three) were aged for 72 hours and brains were stained for the markers indicated. PointedP1 (PntP1) marks type Ii neuroblasts and Ase⁻ young INPs. (F) Summary of the erm-GAL4 expression design in the type II neuroblast lineage. (One thousand-J) Overexpression of klu in immature INPs leads to supernumerary type II neuroblasts. (Grand-I) Larvae overexpressing klu driven by erm-GAL4 were raised at 31°C for 72 hours ALH and brains were stained for the markers indicated. The white dotted line separates the central brain (left) from the optic lobe (right). (J) Average type Ii neuroblasts per brain lobe in larvae of the genotype indicated. 1×, 2× indicate the re-create number of UAS-klu and erm-GAL4 (Iii) transgenes. Error confined indicate s.e.m. Abbreviations and arrows/arrowheads as Fig. i. Scale bars: x μm in A-A‴,D-Eastward‴; five μm in B,C; 20 μm in G-I.

As an alternative, we tested whether overexpression of klu in neuroblasts indirectly leads to increased office of Klu in immature INPs, triggering them to acquire a neuroblast fate. We searched for GAL4 lines that can drive expression of the UAS transgene in immature INPs. The erm-GAL4 transgene inserted on the third chromosome (3) in the fly genome is sufficient to induce UAS transgene expression in INPs merely not in blazon Ii neuroblasts (Pfeiffer et al., 2008; Weng et al., 2010). The identical erm-GAL4 transgene inserted on the second chromosome (II) (kindly provided past Dr 1000. Rubin, HHMI) showed a like spatial expression pattern in larval brain, as ectopic expression of a UAS-prospero transgene driven by erm-GAL4 (Two) induced premature loss of young INPs and INPs without affecting the maintenance of type 2 neuroblasts (supplementary material Fig. S4; northward=8). We adjacent tested whether onset of the erm-GAL4 (II) and (III) activity occurs in young INPs by colocalizing the expression of a UAS-GFP reporter transgene with Ase and PointedP1 (PntP1). We reproducibly detected GFP expression driven by erm-GAL4 (II) in both Ase⁻ and Ase⁺ immature INPs (Fig. 3D-D‴,F; n=viii). By dissimilarity, the reporter expression driven past erm-GAL4 (III) was only commencement detected specifically in Ase⁺ immature INPs (Fig. 3E-F; due north=8). We then tested whether increased role of klu in Ase⁻ or Ase⁺ immature INPs can lead to the formation of supernumerary neuroblasts. Indeed, misexpression of klu driven by erm-GAL4 (II) led to a greater than 10-fold increase in blazon II neuroblasts per brain lobe compared with a similarly staged wild-type encephalon lobe (Fig. 3G,J and Fig. 1F; n=8). Although misexpression of one copy of UAS-klu driven by one re-create of erm-GAL4 (III) failed to induce supernumerary type II neuroblasts, doubling the number of UAS-klu and erm-GAL4 (Three) transgenes led to modest expansion of type II neuroblasts (Fig. 3H-J; n=12 per genotype). Together, these data strongly advise that young INPs can indeed revert to type Two neuroblasts in response to misexpression of klu.

Promotion by Klu of supernumerary type II neuroblast formation is dependent on the zinc-finger motifs

klu, the wing ortholog of the mammalian Wilms tumor i (WT1) gene, encodes a putative transcriptional regulator characterized past iv C₂H₂ zinc-finger motifs in the C-terminus (Klein and Campos-Ortega, 1997; Yang et al., 1997). Vertebrate studies have shown that WT1 requires its zinc-finger motifs to regulate transcription of its target genes (Roberts, 2005). To test whether Klu triggers supernumerary neuroblasts by interim equally a transcriptional regulator, we ectopically expressed a serial of UAS-klu transgenes in neuroblasts (Fig. 4A). We focused our analyses on the type 2 lineage as overexpression of the full-length Klu transgenic protein specifically led to the expansion of type Ii neuroblasts (Fig. 2E-Due east″).

Induction of supernumerary type Two neuroblasts past Klu is dependent on the zinc-finger motifs. (A) The klu transgenes used in this study. (B-D) Drosophila larvae overexpressing various klu transgenes were raised at 31°C for 72 hours ALH and brains were stained for the markers indicated. The white dotted line separates the central brain (left) from the optic lobe (right). Phalloidin (Phall) marks the cell cortex. Type II neuroblasts (Dpn⁺ Ase⁻) are indicated (arrows). Calibration bar: 20 μm. (East) Average blazon Two neuroblasts per brain lobe in larvae of the genotype indicated. Error confined indicate s.east.chiliad.

Expression of the Klu^ane-583 transgenic protein (which lacks all four zinc-finger motifs) failed to induce supernumerary neuroblasts, indicating that the zinc-finger motifs are indispensable for Klu to promote the identity of type II neuroblasts (Fig. 4B,E; 100%, northward=10 per genotype). Although expression of the Klu^Δzf1 transgenic protein (which lacks zinc-finger i) was sufficient to induce supernumerary neuroblasts, it appeared to exist less stiff than expression of full-length Klu (Fig. 2E-E″ and Fig. 4C,E; 100%, n=10 per genotype). This result strongly suggests that zinc-finger 1 is necessary for the optimal office of Klu in promoting the formation of supernumerary neuroblasts. Significantly, expression of the Klu^Δzf4 transgenic poly peptide (which lacks zinc-finger 4) completely failed to induce supernumerary neuroblasts, strongly suggesting that zinc-finger 4 is essential for Klu function (Fig. 4D,E; 100%, n=10).

Finally, we confirmed that expression levels of the various truncated Klu transgenic proteins under the above experimental conditions were indistinguishable from each other (supplementary material Fig. S5). Our data correlate well with a previously published domain assay of the Klu protein in the developing sensory organ precursor cell (Kaspar et al., 2008). Thus, we propose that Klu promotes the identity of type II neuroblasts by regulating factor transcription.

Brat prevents the reversion of immature INPs to type II neuroblasts by antagonizing Klu

Our information thus far are consistent with our hypothesis that Deviling distinguishes an immature INP from its sibling type II neuroblast in office past antagonizing the function of Klu. Nosotros directly tested whether removal of klu function can suppress the germination of supernumerary neuroblasts and restore INPs in deviling^11/k06028 strong hypomorphic mutant brains. The command type II neuroblast clones conveying both copies of the wild-blazon klu gene in brat^11/k06028 mutant brains contained mostly neuroblasts and very few INPs (Fig. 5A-A‴⁗,C; 100%, n=10 clones). By contrast, klu mutant type Two neuroblast clones in brat^11/k06028 mutant brains contained a single neuroblast per clone and possessed INPs and GMCs (Fig. 5B-C; 92%, n=12 clones). These data strongly support our hypothesis that Brat distinguishes an immature INP from its sibling type II neuroblast past antagonizing Klu.

Brat suppresses reversion of immature INPs by antagonizing Klu. (A-C) Removal of klu function suppresses supernumerary type II neuroblasts and restores the formation of INPs and GMCs in brat potent hypomorphic mutant brains. (A-B⁗) brat^11/k06028 mutant Drosophila larvae conveying GFP-marked control (klu ^+/+) and klu mutant type Ii neuroblast mosaic clones (outlined by the xanthous dotted line) were aged for 72 hours after clone induction and brains were stained for the markers indicated. (C) Quantification of diverse cell types in the control and klu mutant clones in brat^{eleven/k06028} mutant brains. (D-F) Co-expression of Brat suppresses Klu-induced supernumerary type Ii neuroblasts. (D-East⁗) Larvae conveying GFP-marked type II neuroblast lineage clones (outlined by the yellow dotted line) overexpressing klu or klu and brat were aged for 72 hours after clone induction and brains were stained for the markers indicated. (F) Average type II neuroblasts per encephalon lobe in larvae of the genotype indicated. Error confined point south.east.thousand. Arrows/arrowheads as Fig. 1. Scale bars: 10 μm.

To ostend that Brat can indeed antagonize Klu in immature INPs, nosotros induced genetic clones derived from a single blazon Ii neuroblast overexpressing klu alone or klu and brat simultaneously. The control clones overexpressing klu consisted of virtually all neuroblasts with very few Ase⁻ immature INPs (Fig. 5D-D⁗,F; 62.5%, n=xvi clones). Co-expression of brat merely not an unrelated UAS transgene significantly suppressed the supernumerary neuroblast phenotype and restored the formation of Ase⁻ and Ase⁺ young INPs, INPs and GMCs in the type Two neuroblast clones overexpressing klu (Fig. 5E-F; 100%, n=10 clones; data not shown). Finally, overexpression of brat alone did not modify cell fate specification in the type II neuroblast clones (information not shown). Together, these data led usa to conclude that Brat antagonizes Klu in the immature INP, distinguishing it from its sibling blazon Ii neuroblast (Fig. 6H).

Abnormal activation of Notch signaling induces reversion of immature INPs through klu . (A-D) Removal of klu function suppresses supernumerary type 2 neuroblasts induced by constitutively activated Notch signaling. (A-C⁗) Drosophila larvae carrying GFP-marked wild-type (klu ^+/+) or klu^−/− type II neuroblast mosaic clones (outlined by the yellow dotted line) overexpressing Notch_intra were aged for 72 hours after clone consecration and brains were stained for the markers indicated. (D) The frequency of clones containing one or more type 2 neuroblasts in larvae of the genotype indicated. (Due east-1000) Overexpression of klu prevents Notch mutant type Two neuroblasts from premature differentiation. (East-F⁗) Larvae carrying GFP-marked Notch mutant type II neuroblast mosaic clones (outlined past the yellow dotted line) lonely or overexpressing klu were anile for 72 hours subsequently clone induction and brains were stained for the markers indicated. (G) The frequency of clones containing i or no type II neuroblasts in larvae of the genotype indicated. (H) Model: Brat or Numb preclude the reversion of immature INPs to type Two neuroblasts by antagonizing Klu. Abbreviations and arrows/arrowheads as Fig. ane. Scale bars: 10 μm.

Abnormal activation of Notch signaling promotes the reversion of immature INPs through klu

The basal protein Numb, which is an evolutionarily conserved inhibitor of Notch signaling, is besides necessary for the formation of INPs in larval encephalon, only how Numb regulates maturation of immature INPs has never been characterized (Rhyu et al., 1994; Guo et al., 1996; Bowman et al., 2008). We first investigated the office of Numb during maturation past assessing the identity of cells in the GFP-marked clones derived from a single numb null mutant type Two neuroblast. Whereas a 24-hour numb mutant clone independent five.9±3.2 neuroblasts, 4.7±one.seven Ase⁻ immature INPs and no Ase⁺ immature INPs, a 72-hour numb clone possessed 195.4±35.four neuroblasts, 122.2±43.6 Ase⁻ young INPs and no Ase⁺ immature INPs (supplementary cloth Fig. S1H and Fig. S6; n=9 per stage). Indistinguishable from the numb mutant clones, the type Ii neuroblast clones expressing a constitutively activated form of Notch (Notch_intra ) also contained neuroblasts and Ase⁻ immature INPs but never Ase⁺ immature INPs and INPs (Fig. 6A-A⁗,D; 100%, n=fifteen). Numb thereby functions to prevent an immature INP from acquiring a neuroblast fate and instead promotes it to assume an INP identity most likely through inhibition of Notch signaling.

We adjacent tested whether aberrant activation of Notch signaling induces the reversion of young INPs to blazon Ii neuroblasts via a Klu-dependent mechanism. Removal of klu function significantly reduced supernumerary neuroblasts and restored INPs and GMCs in half of the clones derived from type Two neuroblasts overexpressing Notch_intra (Fig. 6B-B‴,D; northward=xviii clones). Most significantly, 33.iii% of these clones possessed a single neuroblast per clone (Fig. 6C-D; due north=18 clones). Thus, abnormal activation of Notch signaling in immature INPs leads to the formation of supernumerary neuroblasts via a Klu-dependent machinery.

We directly tested whether klu acts downstream of Notch signaling to maintain type II neuroblasts by assessing the identity of cells in the mosaic clones derived from Notch mutant blazon Ii neuroblasts and those overexpressing klu. Whereas most Notch mutant clones did not contain neuroblasts, overexpression of klu completely suppressed the premature loss of type II neuroblasts in the Notch mutant clones (Fig. 6E-G; 100%, north=8 clones). This issue strongly suggests that Notch signaling maintains the identity of type Ii neuroblasts via a klu-dependent mechanism. Interestingly, overexpression of klu in Notch mutant type Ii neuroblast clones failed to induce the germination of supernumerary neuroblasts (Fig. 6F-Yard; 100%, n=eight clones). Thus, we propose that aberrant activation of Notch signaling induces the reversion of immature INPs to type Two neuroblasts by activating multiple downstream genes including klu (Fig. 6H).

Aberrant activation of Notch signaling promotes reversion of GMCs through klu

Although klu is necessary for the maintenance of type I neuroblasts, overexpression of klu did not lead to an increase in type I neuroblasts. One plausible reason is that additional fate determinants might office redundantly in the specification of GMC identity, leading u.s.a. to identify Notch signaling as an excellent candidate (Bowman et al., 2008; Wirtz-Peitz et al., 2008; Kaspar et al., 2008). We tested this hypothesis by first overexpressing klu in the numb mutant clones. Whereas the numb mutant clones possessed an average of iii neuroblasts per clone, overexpression of klu tripled the number of neuroblasts in the aforementioned genetic background (supplementary fabric Fig. S7; n=10 per genotype). This indicates that increased function of klu can trigger a further increase in supernumerary type I neuroblasts in the absence of Numb.

We adjacent tested whether Klu tin exacerbate the formation of supernumerary type I neuroblasts induced by activated Notch signaling past examining the identity of cells in the clones derived from a unmarried type I neuroblast ectopically expressing Notch_intra alone or Notch_intra and klu simultaneously. Although the type I neuroblast clones overexpressing Notch_intra contained an average of 6 neuroblasts per clone, but 60% of these clones contained more than than ane neuroblast per clone (Fig. 7B-B⁗,D; north=10 per genotype). Past contrast, the blazon I neuroblast clones co-expressing Notch_intra and klu independent an boilerplate of 18 neuroblasts per clone, and 100% of the clones displayed the supernumerary neuroblast phenotype (Fig. 7C-D; north=10 per genotype). Since the clones derived from neuroblasts overexpressing Notch_intra alone or Notch_intra and klu contained GMCs and their progeny, information technology is unlikely that the supernumerary neuroblasts arose from symmetric neuroblast division. Instead, increased function of klu about likely farther enhances the reversion of GMCs to type I neuroblasts induced by aberrant activation of Notch signaling. To test whether activated Notch signaling promotes the reversion of GMCs to type I neuroblasts via a klu-dependent mechanism, nosotros induced type I neuroblast clones overexpressing Notch_intra with or without klu function. Removal of klu function significantly reduced the average number of supernumerary neuroblasts per clone equally well as the frequency of clones containing greater than one neuroblast compared with the command clones (Fig. 7E-1000; northward=xx clones per genotype). Thus, we advise that abnormal activation of Notch signaling induces the reversion of GMCs to type I neuroblasts by activating multiple downstream genes including klu (Fig. 7H).

Abnormal activation of Notch signaling induces reversion of GMCs in role through klu . (A-D) Co-expression of klu further exacerbates the formation of supernumerary blazon I neuroblasts induced by constitutively activated Notch signaling. (A-C⁗) Drosophila larvae carrying GFP-marked type I neuroblast lineage clones (outlined by the yellow dotted line) overexpressing klu, Notch_intra or klu and Notch_intra were aged for 48 hours after clone induction and brains were stained for the markers indicated. (D) Boilerplate type I neuroblasts per clone and the frequency of clones containing one or more type I neuroblasts in larvae of the genotype indicated. (E-G) Removal of klu office suppresses supernumerary type I neuroblasts induced by constitutively activated Notch signaling. (E-F⁗) Larvae conveying GFP-marked klu ^+/+ or klu^−/− type I neuroblast mosaic clones (outlined past the xanthous dotted line) overexpressing Notch_intra were aged for 72 hours later clone induction and brains were stained for the markers indicated. (One thousand) Average type I neuroblasts per clone and the frequency of clones containing i or more type I neuroblasts in larvae of the genotype indicated. (H) Model: Numb prevents the reversion of GMCs to type I neuroblasts by antagonizing Klu. Abbreviations and arrows/arrowheads as Fig. 1. Scale bars: 10 μm.

DISCUSSION

Asymmetric stem cell division provides an efficient mechanism to preserve a steady stem cell pool while generating differentiated progeny inside the tissue where the stem cells reside. Precise spatial command of the stem prison cell determinants inherited by both sibling cells in every disproportionate cell division ensures that a girl cell maintains the stem cell characteristics while the sibling progeny acquires the progenitor jail cell identity. In mitotic type 2 neuroblasts, the basal proteins Brat and Numb segregate into immature INPs and are required for the formation of INPs (Bello et al., 2006; Betschinger et al., 2006; Lee et al., 2006a; Lee et al., 2006c; Wang et al., 2006; Bowman et al., 2008; Wirtz-Peitz et al., 2008). Our written report significantly extends the findings from previous studies and showed that Deviling and Numb part in young INPs to preclude them from acquiring a neuroblast fate while promoting the INP identity (supplementary textile Figs S1, S6). Identification and label of the klu gene led united states of america to advise that Brat and Numb converge to exert precise control of Klu to distinguish an immature INP from its sibling type II neuroblast (Fig. 6H). Numb besides prevents a GMC from reverting to a type I neuroblast by inhibiting Notch signaling in the type I neuroblast lineage (Fig. seven and supplementary material Fig. S7). Interestingly, although overexpression of klu was bereft to induce supernumerary type I neuroblasts, increased function of klu can drastically enhance the reversion of GMCs to type I neuroblasts in the presence of activated Notch signaling (Fig. vii). Thus, we propose that abnormal activation of Notch signaling induces reversion of GMCs by activating multiple downstream genes including klu. Together, our information led us to conclude that precise regulation of klu by multiple signaling mechanisms distinguishes a progenitor cell from its sibling stalk cell during asymmetric stem cell division.

Regulation of INP maturation

The essential role of Brat and Numb in regulating the germination of INPs is well established, but lack of insight into maturation has hindered investigation into the mechanisms by which these ii proteins distinguish an immature INP from its sibling type II neuroblast (Bello et al., 2006; Betschinger et al., 2006; Lee et al., 2006a; Lee et al., 2006c; Wang et al., 2006; Bowman et al., 2008; Wirtz-Peitz et al., 2008). A previous written report divers young INPs by the following criteria: (1) being immediately next to the parental type II neuroblast, (2) lacking Dpn expression and (three) displaying a very low level of CycE expression (Bowman et al., 2008). Based on these criteria, analyses of the spatial expression pattern of various cell fate markers in the type II neuroblast lineage clones in wild-type brains revealed that onset of Ase expression correlates with an intermediate stage of maturation (supplementary cloth Fig. S1A-A⁗). In the xvi-hour clones, we reproducibly observed one type Two neuroblast (Dpn⁺ Ase⁻ CycE⁺), 2 to iii Ase⁻ immature INPs (Dpn⁻ Ase⁻ CycE⁻), two to iii Ase⁺ immature INPs (Dpn⁻ Ase⁺ CycE⁻) and INPs (Dpn⁺ Ase⁺ CycE⁺) (supplementary material Fig. S1A-B). Furthermore, we showed that Ase⁻ young INPs maintain expression of the type II neuroblast-specific mark PntP1, whereas Ase⁺ immature INPs showed virtually undetectable PntP1 expression (Fig. 3F-H). Thus, onset of Ase expression should serve as a useful marker for an intermediate stage during maturation.

Our data led u.s. to suggest that Brat distinguishes an immature INP from its sibling type II neuroblast by indirectly antagonizing the office of Klu based on the post-obit evidence. Kickoff, Klu was undetectable in Ase⁻ young INPs in the brat single-mutant or deviling and numb double-mutant type II neuroblast clones (data not shown). Thus, a Brat-independent mechanism must exist to downregulate Klu in immature INPs. Second, overexpression of a truncated Deviling transgenic poly peptide lacking the NHL domain, which is required for repression of mRNA translation (Sonoda and Wharton, 2001), completely suppresses the formation of supernumerary neuroblasts (H.Chiliad. and C.-Y.L., unpublished). Thus, it is unlikely that downregulation of Klu in immature INPs occurs via a Brat-dependent translational repression of klu mRNA. We propose that Deviling might suppress the expression of a co-cistron necessary for the function of Klu, simply as WT1 requires co-factors in order to regulate the expression of its target genes in vertebrates (Roberts, 2005). Further investigation will exist necessary to discern how Brat establishes restricted developmental potential in young INPs by antagonizing the office of Klu.

The part of Klu in promoting neuroblast identity

WT1 requires its zinc-finger motifs to regulate transcription of its target genes and can function as an activator or a repressor of transcription in a context-dependent manner (Roberts, 2005). A previous study showed that overexpression of Klu tin can partially suppress the expression of a lacZ reporter transgene containing the cis-regulatory elements from the fifty-fifty-skipped gene, a putative direct target of Klu, in the fly embryonic central nervous system (McDonald et al., 2003). Since Klu and WT1 display extensive homology in zinc-fingers two-4, Klu is likely to recognize a similar Dna binding sequence every bit WT1 (Klein and Campos-Ortega, 1997; Yang et al., 1997; McDonald et al., 2003). The even-skipped cis-regulatory element contains three putative WT1 binding sites, only nucleotide substitutions in these sites that were predicted to cancel Klu binding failed to render the lacZ reporter transgene unresponsive to overexpression of klu (McDonald et al., 2003). These data led usa to speculate that Klu might recognize a singled-out consensus DNA binding sequence to WT1. To exam this hypothesis, we generated two UAS-WT1 transgenes that encode the ii virtually prevalent isoforms of the WT1 protein, WT1 −KTS and WT1 +KTS. Interestingly, neither WT1 transgene, when overexpressed by wor-GAL4, triggered the formation of supernumerary type II neuroblasts in larval brain (data non shown). This is consistent with Klu recognizing a distinct consensus Deoxyribonucleic acid bounden sequence to WT1. Yet, we cannot dominion out the possibility that the inability of the WT1 transgenic protein to induce supernumerary type 2 neuroblasts is simply due to the absenteeism of necessary co-factors in the wing, as repression of target cistron transcription past WT1 requires additional co-factors in vertebrates (Shervington et al., 2006). More than studies will exist necessary to elucidate the molecular function of Klu in promoting type II neuroblast identity.

Progressive brake of developmental potential during maturation of young INPs

Restricted developmental potential functionally defines progenitor cells and allows them to generate differentiated progeny through limited rounds of cell division without impinging on the homeostatic state of the stem cell pool (Zon, 2008; Knoblich, 2010; Weng and Lee, 2011). Despite their importance, the molecular mechanisms by which progenitor cells acquire restricted developmental potential remain experimentally inaccessible in most stem prison cell lineages. Even so, studies from diverse groups accept paved the fashion for using fly larval encephalon neuroblast lineages every bit an in vivo model organisation for investigating how progenitor cells acquire restricted developmental potential (Bello et al., 2008; Boone and Doe, 2008; Bowman et al., 2008; Bayraktar et al., 2010; Weng et al., 2010).

In this study, we draw the expression design of boosted molecular markers that allow us to unambiguously identify 2 distinct populations of immature INPs. Furthermore, we provide experimental prove strongly suggesting that these ii groups of young INPs possess singled-out functional properties. More specifically, Ase⁻ immature INPs readily revert to type II neuroblasts in response to misexpression of Klu, whereas Ase⁺ immature INPs appear much less responsive to Klu. These data led us to suggest that the genome in immature INPs becomes reprogrammed during maturation such that these cells become progressively less responsive to neuroblast fate determinants such as Klu. Equally a consequence, an INP becomes completely unresponsive to Klu following maturation. Farther experiments will be required to validate this model in the time to come.

Supplementary Material

Acknowledgements

We thank Drs C. Doe, A. Gould, T. Klein, T. Orr-Weaver, 1000. Rubin, J. Skeath, G. Struhl and X. Yang for wing stocks and antibody reagents; Dr M. Mindren for WT1 cDNA clones; the Bloomington Drosophila Stock Center and the Developmental Studies Hybridoma Bank for antibodies; Krista Fifty. Golden for technical help throughout the course of this work; and the members of the C.-Y.L. laboratory for reading the manuscript and providing disquisitional comments.

Footnotes

Funding

H.1000. was supported by a fellowship from the Japan Club for the Promotion of Science. C.-Y.L. is supported by the Academy of Michigan Starting time-Upwardly Fund, the Burroughs Wellcome Fund Career Accolade in the Biomedical Sciences [1006160.01], a Sontag Foundation Distinguished Scientist Award and the National Institutes of Health [R01-GM092818;, R01-NS077914]. Deposited in PMC for release after 12 months.

Competing interests statement

The authors declare no competing financial interests.

Supplementary material

Supplementary cloth available online at http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.081687/-/DC1

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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3392700/

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