Progenitor properties of symmetrically dividing Drosophila neuroblasts during embryonic and larval development

Asymmetric cell division generates two daughter cells of differential gene expression and/or cell shape. Drosophila neuroblasts undergo typical asymmetric divisions with regard to both features; this is achieved by asymmetric segregation of cell fate determinants (such as Prospero) and also by asymmetric spindle formation. The loss of genes involved in these individual asymmetric processes has revealed the roles of each asymmetric feature in neurogenesis, yet little is known about the fate of the neuroblast progeny when asymmetric processes are blocked and the cells divide symmetrically. We genetically created such neuroblasts, and found that in embryos, they were initially mitotic and then gradually differentiated into neurons, frequently forming a clone of cells homogeneous in temporal identity. By contrast, larval neuroblasts with the same genotype continued to proliferate without differentiation. Our results indicate that asymmetric divisions govern lineage length and progeny fate, consequently generating neural diversity, while the progeny fate of symmetrically dividing neuroblasts depends on developmental stages, presumably reflecting differential activities of Prospero in the nucleus.     

Cortical aPKC kinase activity distinguishes neural stem cells from progenitor cells by ensuring asymmetric segregation of Numb

During asymmetric stem cell division, polarization of the cell cortex targets fate determinants unequally into the sibling daughters, leading to regeneration of a stem cell and production of a progenitor cell with restricted developmental potential. In mitotic neural stem cells (neuroblasts) in fly larval brains, the antagonistic interaction between the polarity proteins Lethal (2) giant larvae (Lgl) and atypical Protein Kinase C (aPKC) ensures self-renewal of a daughter neuroblast and generation of a progenitor cell by regulating asymmetric segregation of fate determinants. In the absence of lgl function, elevated cortical aPKC kinase activity perturbs unequal partitioning of the fate determinants including Numb and induces supernumerary neuroblasts in larval brains. However, whether increased aPKC function triggers formation of excess neuroblasts by inactivating Numb remains controversial. To investigate how increased cortical aPKC function induces formation of excess neuroblasts, we analyzed the fate of cells in neuroblast lineage clones in lgl mutant brains. Surprisingly, our analyses revealed that neuroblasts in lgl mutant brains undergo asymmetric division to produce progenitor cells, which then revert back into neuroblasts. In lgl mutant brains, Numb remained localized in the cortex of mitotic neuroblasts and failed to segregate exclusively into the progenitor cell following completion of asymmetric division. These results led us to propose that elevated aPKC function in the cortex of mitotic neuroblasts reduces the function of Numb in the future progenitor cells. We identified that the acyl-CoA binding domain containing 3 protein (ACBD3) binding region is essential for asymmetric segregation of Numb in mitotic neuroblasts and suppression of the supernumerary neuroblast phenotype induced by increased aPKC function. The ACBD3 binding region of Numb harbors two aPKC phosphorylation sites, serines 48 and 52. Surprisingly, while the phosphorylation status at these two sites directly impinged on asymmetric segregation of Numb in mitotic neuroblasts, both the phosphomimetic and non-phosphorylatable forms of Numb suppressed formation of excess neuroblasts triggered by increased cortical aPKC function. Thus, we propose that precise regulation of cortical aPKC kinase activity distinguishes the sibling cell identity in part by ensuring asymmetric partitioning of Numb into the future progenitor cell where Numb maintains restricted potential independently of regulation by aPKC.     

Asymmetric centrosome behavior and the mechanisms of stem cell division

The ability of dividing cells to produce daughters with different fates is an important developmental mechanism conserved from bacteria to fungi, plants, and metazoan animals. Asymmetric outcomes of a cell division can be specified by two general mechanisms: asymmetric segregation of intrinsic fate determinants or asymmetric placement of daughter cells into microenvironments that provide extrinsic signals that direct cells to different states. For both, spindle orientation must be coordinated with the localization of intrinsic determinants or source of extrinsic signals to achieve the proper asymmetric outcome. Recent work on spindle orientation in Drosophila melanogaster male germline stem cells and neuroblasts has brought into sharp focus the key role of differential centrosome behavior in developmentally programmed asymmetric division (for reviews see Cabernard, C., and C.Q. Doe. 2007. Curr. Biol. 17:R465-R467; Gonzalez, C. 2007. Nat. Rev. Genet. 8:462-472). These findings provide new insights and suggest intriguing new models for how cells coordinate spindle orientation with their cellular microenvironment to regulate and direct cell fate decisions within tissues.     

Molecular mechanisms of asymmetric divisions in mammary stem cells

Stem cells have the remarkable ability to undergo proliferative symmetric divisions and self‐renewing asymmetric divisions. Balancing of the two modes of division sustains tissue morphogenesis and homeostasis. Asymmetric divisions of Drosophila neuroblasts (NBs) and sensory organ precursor (SOP) cells served as prototypes to learn what we consider now principles of asymmetric mitoses. They also provide initial evidence supporting the notion that aberrant symmetric divisions of stem cells could correlate with malignancy. However, transferring the molecular knowledge of circuits underlying asymmetry from flies to mammals has proven more challenging than expected. Several experimental approaches have been used to define asymmetry in mammalian systems, based on daughter cell fate, unequal partitioning of determinants and niche contacts, or proliferative potential. In this review, we aim to provide a critical evaluation of the assays used to establish the stem cell mode of division, with a particular focus on the mammary gland system. In this context, we will discuss the genetic alterations that impinge on the modality of stem cell division and their role in breast cancer development.     

Characterization of histone inheritance patterns in the Drosophila female germline

Stem cells have the unique ability to undergo asymmetric division which produces two daughter cells that are genetically identical, but commit to different cell fates. The loss of this balanced asymmetric outcome can lead to many diseases, including cancer and tissue dystrophy. Understanding this tightly regulated process is crucial in developing methods to treat these abnormalities. Here, we report that during a Drosophila female germline stem cell asymmetric division, the two daughter cells differentially inherit histones at key genes related to either maintaining the stem cell state or promoting differentiation, but not at constitutively active or silenced genes. We combine histone labeling with DNA Oligopaints to distinguish old versus new histones and visualize their inheritance patterns at a single‐gene resolution in asymmetrically dividing cells in vivo. This strategy can be applied to other biological systems involving cell fate change during development or tissue homeostasis in multicellular organisms.     

Polarization of Drosophila Neuroblasts During Asymmetric Division

During Drosophila development, neuroblasts divide to generate progeny with two different fates. One daughter cell self-renews to maintain the neuroblast pool, whereas the other differentiates to populate the central nervous system. The difference in fate arises from the asymmetric distribution of proteins that specify either self-renewal or differentiation, which is brought about by their polarization into separate apical and basal cortical domains during mitosis. Neuroblast symmetry breaking is regulated by numerous proteins, many of which have only recently been discovered. The atypical protein kinase C (aPKC) is a broad regulator of polarity that localizes to the neuroblast apical cortical region and directs the polarization of the basal domain. Recent work suggests that polarity can be explained in large part by the mechanisms that restrict aPKC activity to the apical domain and those that couple asymmetric aPKC activity to the polarization of downstream factors. Polarized aPKC activity is created by a network of regulatory molecules, including Bazooka/Par-3, Cdc42, and the tumor suppressor Lgl, which represses basal recruitment. Direct phosphorylation by aPKC leads to cortical release of basal domain factors, preventing them from occupying the apical domain. In this framework, neuroblast polarity arises from a complex system that orchestrates robust aPKC polarity, which in turn polarizes substrates by coupling phosphorylation to cortical release.Cells use polarity for remarkably diverse functions. In this article, I discuss a polarity that is harnessed to generate daughter cells with different fates. Using polarity to divide asymmetrically addresses several challenges that complex organisms face. The diversification of cell types and tissues that occurs during the development of complex organisms is one such challenge. Drosophila neuroblasts, the subject of this article, undergo repeated symmetry breaking asymmetric cell divisions (ACDs) to populate the central nervous system. In a similar manner in adult organisms, ACDs are important for adult homeostasis, replenishing cells that are turned over during the course of normal physiology (Betschinger and Knoblich 2004).A fundamental aspect of ACD is the production of daughter cells containing distinct fate determinants. To segregate fate determinants, the cell becomes polarized to form mutually exclusive cortical domains, each with a set of fate determinants appropriate for one of the two daughter cells. The cleavage furrow forms at the interface of the two domains, partitioning the fate determinants into the two daughter cells where they function to either self-renew (to keep the progenitor population) or to differentiate (e.g., by changing the pattern of gene expression). One of the unique features of the symmetry breaking that occurs during ACD, at least as implemented by the neuroblast, is that it is remarkably dynamic, developing early in mitosis and depolarizing following the completion of cytokinesis.Since the discovery of the first polarized components, neuroblasts have been an excellent model system for investigating the mechanisms of cell polarization and have been extensively analyzed. Although aspects of neuroblast polarity remain unclear, a core framework for how polarity is created and maintained is emerging. In this article, I focus on neuroblast polarity as centered around the activity of atypical protein kinase C, which has emerged as a key regulator of the process. In this framework, neuroblast polarity can be explained by events that polarize aPKC and those that couple aPKC activity to the polarization of fate determinants.     

Roles for polarity and nuclear determinants in specifying daughter cell fates after an asymmetric cell division in the maize leaf

Asymmetric cell divisions occur repeatedly during plant development, but the mechanisms by which daughter cells are directed to adopt different fates are not well understood [1,2]. Previous studies have demonstrated roles for positional information in specification of daughter cell fates following asymmetric divisions in the embryo [3] and root [4]. Unequally inherited cytoplasmic determinants have also been proposed to specify daughter cell fates after some asymmetric cell divisions in plants [1,2,5], but direct evidence is lacking. Here we investigate the requirements for specification of stomatal subsidiary cell fate in the maize leaf by analyzing four mutants disrupting the asymmetric divisions of subsidiary mother cells (SMCs). We show that subsidiary cell fate does not depend on proper localization of the new cell wall during the SMC division, and is not specified by positional information acting on daughter cells after completion of the division. Instead, our data suggest that specification of subsidiary cell fate depends on polarization of SMCs and on inheritance of the appropriate daughter nucleus. We thus provide evidence of a role for unequal inheritance of an intracellular determinant in specification of cell fate after an asymmetric plant cell division.     

Where does asymmetry come from? Illustrating principles of polarity and asymmetry establishment in Drosophila neuroblasts

Asymmetric cell division (ACD) is the fundamental process through which one cell divides into two cells with different fates. In animals, it is crucial for the generation of cell-type diversity and for stem cells, which use ACD both to self-renew and produce one differentiating daughter cell. One of the most prominent model systems of ACD, Drosophila neuroblasts, relies on the PAR complex, a conserved set of proteins governing cell polarity in animals. Here, we focus on recent advances in our understanding of the mechanisms that control the orientation of the neuroblast polarity axis, how the PAR complex is positioned, and how its activity may regulate division orientation and cell fate determinant localization and discuss how important findings about the composition polarity complexes in other models may apply to neuroblasts.     

Lethal giant larvae controls the localization of notch-signaling regulators numb, neuralized, and Sanpodo in Drosophila sensory-organ precursor cells

Asymmetric distribution of fate determinants is a fundamental mechanism underlying the acquisition of distinct cell fates during asymmetric division. In Drosophila neuroblasts, the apical DmPar6/DaPKC complex inhibits Lethal giant larvae (Lgl) to promote the basal localization of fate determinants. In contrast, in the sensory precursor (pI) cells that divide asymmetrically with a planar polarity, Lgl inhibits Notch signaling in the anterior pI daughter cell, pIIb, by a yet-unknown mechanism. We show here that Lgl promotes the cortical recruitment of Partner of Numb (Pon) and regulates the asymmetric distribution of the fate determinants Numb and Neuralized during the pI cell division. Analysis of Pon-GFP and Histone2B-mRFP distribution in two-color movies confirmed that Lgl regulates Pon localization. Moreover, posterior DaPKC restricts Lgl function to the anterior cortex at mitosis. Thus, Lgl functions similarly in neuroblasts and in pI cells. We also show that Lgl promotes the acquisition of the pIIb cell fate by inhibiting the plasma membrane localization of Sanpodo and thereby preventing the activation of Notch signaling in the anterior pI daughter cell. Thus, Lgl regulates cell fate by controlling Pon cortical localization, asymmetric localization of Numb and Neuralized, and plasma-membrane localization of Sandopo.     

The role of asymmetric cell division in pteridophyte cell differentiation. I. Localized metal accumulation and differentiation inVittaria gemmae andOnoclea prothallia

Summary In gemmae ofVittaria graminifolia and prothallia ofOnoclea sensibilis, cell differentiation is initiated by nuclear migration and geometrically asymmetric cell division. The small daughter cells inVittaria develop into antheridia in the presence of gibberellic acid or into rhizoids or new prothallia in its absence. Antheridial differentiation from asymmetric division is induced inOnoclea byPteridium antheridiogen, whereas rhizoid or vegetative cell formation occurs in its absence. Although asymmetric cytokinesis initiates differentiation, it does not in itself determine the developmental fate of the smaller cell. Several histochemical techniques demonstrate that prior to nuclear migration and cell division, Ca²⁺ accumulates in the cytoplasm and wall of the cell at the site where asymmetric division will occur, regardless of the developmental fate of the small cell. The cytoplasmic localization of Ca²⁺ appears to reflect a mobilization of Ca²⁺ from within the cell that eventually moves into the cell wall. We propose that this internal accumulation of Ca²⁺ leads to a localized decrease in cytosolic [Ca²⁺] which in turn may regulate developmental events such as nuclear migration.Publishing prior to 1984 as Alix R. Bassel.     

Polarity in Stem Cell Division: Asymmetric Stem Cell Division in Tissue Homeostasis

Many adult stem cells divide asymmetrically to balance self-renewal and differentiation, thereby maintaining tissue homeostasis. Asymmetric stem cell divisions depend on asymmetric cell architecture (i.e., cell polarity) within the cell and/or the cellular environment. In particular, as residents of the tissues they sustain, stem cells are inevitably placed in the context of the tissue architecture. Indeed, many stem cells are polarized within their microenvironment, or the stem cell niche, and their asymmetric division relies on their relationship with the microenvironment. Here, we review asymmetric stem cell divisions in the context of the stem cell niche with a focus on Drosophila germ line stem cells, where the nature of niche-dependent asymmetric stem cell division is well characterized.Asymmetric cell division allows stem cells to self-renew and produce another cell that undergoes differentiation, thus providing a simple method for tissue homeostasis. Stem cell self-renewal refers to the daughter(s) of stem cell division maintaining all stem cell characteristics, including proliferation capacity, maintenance of the undifferentiated state, and the capability to produce daughter cells that undergo differentiation. A failure to maintain the correct stem cell number has been speculated to lead to tumorigenesis/tissue hyperplasia via stem cell hyperproliferation or tissue degeneration/aging via a reduction in stem cell number or activity (Morrison and Kimble 2006; Rando 2006). This necessity changes during development. The stem cell pool requires expansion earlier in development, whereas maintenance is needed later to sustain tissue homeostasis.There are two major mechanisms to sustain a fixed number of adult stem cells: stem cell niche and asymmetric stem cell division, which are not mutually exclusive. Stem cell niche is a microenvironment in which stem cells reside, and provides essential signals required for stem cell identity (Fig. 1A). Physical limitation of niche “space” can therefore define stem cell number within a tissue. Within such a niche, many stem cells divide asymmetrically, giving rise to one stem cell and one differentiating cell, by placing one daughter inside and another outside of the niche, respectively (Fig. 1A). Nevertheless, some stem cells divide asymmetrically, apparently without the niche. For example, in Drosophila neuroblasts, cell-intrinsic fate determinants are polarized within a dividing cell, and subsequent partitioning of such fate determinants into daughter cells in an asymmetric manner results in asymmetric stem cell division (Fig. 1B) (see Fig. 3A and Prehoda 2009).Open in a separate windowFigure 1.Mechanisms of asymmetric stem cell division. (A) Asymmetric stem cell division by extrinsic fate determinants (i.e., the stem cell niche). The two daughters of stem cell division will be placed in distinct cellular environments either inside or outside the stem cell niche, leading to asymmetric fate choice. (B) Asymmetric stem cell division by intrinsic fate determinants. Fate determinants are polarized in the dividing stem cells, which are subsequently partitioned into two daughter cells unequally, thus making the division asymmetrical. Self-renewing (red line) and/or differentiation promoting (green line) factors may be involved.In this review, we focus primarily on asymmetric stem cell divisions in the Drosophila germ line as the most intensively studied examples of niche-dependent asymmetric stem cell division. We also discuss some examples of stem cell division outside Drosophila, where stem cells are known to divide asymmetrically or in a niche-dependent manner.     

On the roles of Notch, Delta, kuzbanian, and inscuteable during the development of Drosophila embryonic neuroblast lineages

The generation of cellular diversity in the nervous system involves the mechanism of asymmetric cell division. Besides an array of molecules, including the Par protein cassette, a heterotrimeric G protein signalling complex, Inscuteable plays a major role in controlling asymmetric cell division, which ultimately leads to differential activation of the Notch signalling pathway and correct specification of the two daughter cells. In this context, Notch is required to be active in one sibling and inactive in the other. Here, we investigated the requirement of genes previously known to play key roles in sibling cell fate specification such as members of the Notch signalling pathway, e.g., Notch (N), Delta (Dl), and kuzbanian (kuz) and a crucial regulator of asymmetric cell division, inscuteable (insc) throughout lineage progression of 4 neuroblasts (NB1-1, MP2, NB4-2, and NB7-1). Notch-mediated cell fate specification defects were cell-autonomous and were observed in all neuroblast lineages even in cells born from late ganglion mother cells (GMC) within the lineages. We also show that Dl functions non-autonomously during NB lineage progression and clonal cells do not require Dl from within the clone. This suggests that within a NB lineage Dl is dispensable for sibling cell fate specification. Furthermore, we provide evidence that kuz is involved in sibling cell fate specification in the central nervous system. It is cell-autonomously required in the same postmitotic cells which also depend on Notch function. This indicates that KUZ is required to facilitate a functional Notch signal in the Notch-dependent cell for correct cell fate specification. Finally, we show that three neuroblast lineages (NB1-1, NB4-2, and NB7-1) require insc function for sibling cell fate specification in cells born from early GMCs whereas insc is not required in cells born from later GMCs of the same lineages. Thus, there is differential requirement for insc for cell fate specification depending on the stage of lineage progression of NBs.     

The role of centrosomes and astral microtubules during asymmetric division of Drosophila neuroblasts

Drosophila neuroblasts are stem cells that divide asymmetrically to produce another large neuroblast and a smaller ganglion mother cell (GMC). During neuroblast division, several cell fate determinants, such as Miranda, Prospero and Numb, are preferentially segregated into the GMC, ensuring its correct developmental fate. The accurate segregation of these determinants relies on proper orientation of the mitotic spindle within the dividing neuroblast, and on the correct positioning of the cleavage plane. In this study we have analyzed the role of centrosomes and astral microtubules in neuroblast spindle orientation and cytokinesis. We examined neuroblast division in asterless (asl) mutants, which, although devoid of functional centrosomes and astral microtubules, form well-focused anastral spindles that undergo anaphase and telophase. We show that asl neuroblasts assemble a normal cytokinetic ring around the central spindle midzone and undergo unequal cytokinesis. Thus, astral microtubules are not required for either signaling or positioning cytokinesis in Drosophila neuroblasts. Our results indicate that the cleavage plane is dictated by the positioning of the central spindle midzone within the cell, and suggest a model on how the central spindle attains an asymmetric position during neuroblast mitosis. We have also analyzed the localization of Miranda during mitotic division of asl neuroblasts. This protein accumulates in morphologically regular cortical crescents but these crescents are mislocalized with respect to the spindle orientation. This suggests that astral microtubules mediate proper spindle rotation during neuroblast division.     

The Arabidopsis Receptor Kinase ZAR1 Is Required for Zygote Asymmetric Division and Its Daughter Cell Fate

Asymmetric division of zygote is critical for pattern formation during early embryogenesis in plants and animals. It requires integration of the intrinsic and extrinsic cues prior to and/or after fertilization. How these cues are translated into developmental signals is poorly understood. Here through genetic screen for mutations affecting early embryogenesis, we identified an Arabidopsis mutant, zygotic arrest 1 (zar1), in which zygote asymmetric division and the cell fate of its daughter cells were impaired. ZAR1 encodes a member of the RLK/Pelle kinase family. We demonstrated that ZAR1 physically interacts with Calmodulin and the heterotrimeric G protein Gβ, and ZAR1 kinase is activated by their binding as well. ZAR1 is specifically expressed micropylarly in the embryo sac at eight-nucleate stage and then in central cell, egg cell and synergids in the mature embryo sac. After fertilization, ZAR1 is accumulated in zygote and endosperm. The disruption of ZAR1 and AGB1 results in short basal cell and an apical cell with basal cell fate. These data suggest that ZAR1 functions as a membrane integrator for extrinsic cues, Ca²⁺ signal and G protein signaling to regulate the division of zygote and the cell fate of its daughter cells in Arabidopsis.     

The Drosophila NuMA Homolog Mud regulates spindle orientation in asymmetric cell division

During asymmetric cell division, the mitotic spindle must be properly oriented to ensure the asymmetric segregation of cell fate determinants into only one of the two daughter cells. In Drosophila neuroblasts, spindle orientation requires heterotrimeric G proteins and the G alpha binding partner Pins, but how the Pins-G alphai complex interacts with the mitotic spindle is unclear. Here, we show that Pins binds directly to the microtubule binding protein Mud, the Drosophila homolog of NuMA. Like NuMA, Mud can bind to microtubules and enhance microtubule polymerization. In the absence of Mud, mitotic spindles in Drosophila neuroblasts fail to align with the polarity axis. This can lead to symmetric segregation of the cell fate determinants Brat and Prospero, resulting in the mis-specification of daughter cell fates and tumor-like over proliferation in the Drosophila nervous system. Our data suggest a model in which asymmetrically localized Pins-G alphai complexes regulate spindle orientation by directly binding to Mud.     

Numb-Associated Kinase Interacts with the Phosphotyrosine Binding Domain of Numb and Antagonizes the Function of Numb In Vivo

During asymmetric cell division, the membrane-associated Numb protein localizes to a crescent in the mitotic progenitor and is segregated predominantly to one of the two daughter cells. We have identified a putative serine/threonine kinase, Numb-associated kinase (Nak), which interacts physically with the phosphotyrosine binding (PTB) domain of Numb. The PTB domains of Shc and insulin receptor substrate bind to an NPXY motif which is not present in the region of Nak that interacts with Numb PTB domain. We found that the Numb PTB domain but not the Shc PTB domain interacts with Nak through a peptide of 11 amino acids, implicating a novel and specific protein-protein interaction. Overexpression of Nak in the sensory organs causes both daughters of a normally asymmetric cell division to adopt the same cell fate, a transformation similar to the loss of numb function phenotype and opposite the cell fate transformation caused by overexpression of Numb. The frequency of cell fate transformation is sensitive to the numb gene dosage, as expected from the physical interaction between Nak and Numb. These findings indicate that Nak may play a role in cell fate determination during asymmetric cell divisions.     

A mosaic genetic screen for novel mutations affecting Drosophila neuroblast divisions

Background

The asymmetric segregation of determinants during cell division is a fundamental mechanism for generating cell fate diversity during development. In Drosophila, neural precursors (neuroblasts) divide in a stem cell-like manner generating a larger apical neuroblast and a smaller basal ganglion mother cell. The cell fate determinant Prospero and its adapter protein Miranda are asymmetrically localized to the basal cortex of the dividing neuroblast and segregated into the GMC upon cytokinesis. Previous screens to identify components of the asymmetric division machinery have concentrated on embryonic phenotypes. However, such screens are reaching saturation and are limited in that the maternal contribution of many genes can mask the effects of zygotic loss of function, and other approaches will be necessary to identify further genes involved in neuroblast asymmetric division.

Results

We have performed a genetic screen in the third instar larval brain using the basal localization of Miranda as a marker for neuroblast asymmetry. In addition to the examination of pupal lethal mutations, we have employed the MARCM (Mosaic Analysis with a Repressible Cell Marker) system to generate postembryonic clones of mutations with an early lethal phase. We have screened a total of 2,300 mutagenized chromosomes and isolated alleles affecting cell fate, the localization of basal determinants or the orientation of the mitotic spindle. We have also identified a number of complementation groups exhibiting defects in cell cycle progression and cytokinesis, including both novel genes and new alleles of known components of these processes.

Conclusion

We have identified four mutations which affect the process of neuroblast asymmetric division. One of these, mapping to the imaginal discs arrested locus, suggests a novel role for the anaphase promoting complex/cyclosome (APC/C) in the targeting of determinants to the basal cortex. The identification and analysis of the remaining mutations will further advance our understanding of the process of asymmetric cell division. We have also isolated a number of mutations affecting cell division which will complement the functional genomics approaches to this process being employed by other laboratories. Taken together, these results demonstrate the value of mosaic screens in the identification of genes involved in neuroblast division.     

Clueless regulates aPKC activity and promotes self-renewal cell fate in Drosophila lgl mutant larval brains

Asymmetric cell division of Drosophila neural stem cells or neuroblasts is an important process which gives rise to two different daughter cells, one of which is the stem cell itself and the other, a committed or differentiated daughter cell. During neuroblast asymmetric division, atypical Protein Kinase C (aPKC) activity is tightly regulated; aberrant levels of activity could result in tumorigenesis in third instar larval brain. We identified clueless (clu), a genetic interactor of parkin (park), as a novel regulator of aPKC activity. It preferentially binds to the aPKC/Bazooka/Partition Defective 6 complex and stabilizes aPKC levels. In clu mutants, Miranda (Mira) and Numb are mislocalized in small percentages of dividing neuroblasts. Adult mutants are short-lived with severe locomotion defects. Clu promotes tumorigenesis caused by loss of function of lethal(2) giant larvae (lgl) in the larval brain. Removal of clu in lgl mutants rescues Mira and Numb mislocalization and restores the enlarged brain size. Western blot analyses indicate that the rescue is due to the down-regulation of aPKC levels in the lgl clu double mutant. Interestingly, the phenotype of the park mutant, which causes Parkinson's Disease-like symptoms in adult flies, is reminiscent of that of clu in neuroblast asymmetric division. Our study provides the first clue for the potential missing pathological link between temporally separated neurogenesis and neurodegeneration events; the minor defects during early neurogenesis could be a susceptible factor contributing to neurodegenerative diseases at later stages of life.