Dlg,Scrib and Lgl regulate neuroblast cell size and mitotic spindle asymmetry

Asymmetric cell division is important in generating cell diversity from bacteria to mammals. Drosophila melanogaster neuroblasts are a useful model system for investigating asymmetric cell division because they establish distinct apical-basal cortical domains, have an asymmetric mitotic spindle aligned along the apical-basal axis, and divide unequally to produce a large apical neuroblast and a small basal daughter cell (GMC). Here we show that Discs large (Dlg), Scribble (Scrib) and Lethal giant larvae (Lgl) tumour suppressor proteins regulate multiple aspects of neuroblast asymmetric cell division. Dlg/Scrib/Lgl proteins show apical cortical enrichment at prophase/metaphase, and then have a uniform cortical distribution. Mutants have defects in basal protein targeting, a reduced apical cortical domain and reduced apical spindle size. Defects in apical cell and spindle pole size result in symmetric or inverted neuroblast cell divisions. Inverted divisions correlate with the appearance of abnormally small neuroblasts and large GMCs, showing that neuroblast/GMC identity is more tightly linked to cortical determinants than cell size. We conclude that Dlg/Scrib/Lgl are important in regulating cortical polarity, cell size asymmetry and mitotic spindle asymmetry in Drosophila neuroblasts.     

Cortical domains and the mechanisms of asymmetric cell division

Asymmetric cell divisions are central to the generation of cell-fate diversity because factors that are present in a mother cell and distributed unequally at cell division can generate distinct daughters. The process o f asymmetric cell division can be described as consisting of three steps: setting up an asymmetric cue in the mother cell, localizing factors with respect to this cue, and positioning the plane o f cell division so that localized factors are partitioned asymmetrically between daughters. This review describes how specialized cortical domains play a key role in each of these steps and discusses our current understanding of the molecular nature o f cortical domains and the mechanisms by which they may orchestrate asymmetric cell divisions.     

Upregulation of Mitimere and Nubbin acts through cyclin E to confer self-renewing asymmetric division potential to neural precursor cells

In the Drosophila CNS, neuroblasts undergo self-renewing asymmetric divisions, whereas their progeny, ganglion mother cells (GMCs), divide asymmetrically to generate terminal postmitotic neurons. It is not known whether GMCs have the potential to undergo self-renewing asymmetric divisions. It is also not known how precursor cells undergo self-renewing asymmetric divisions. Here, we report that maintaining high levels of Mitimere or Nubbin, two POU proteins, in a GMC causes it to undergo self-renewing asymmetric divisions. These asymmetric divisions are due to upregulation of Cyclin E in late GMC and its unequal distribution between two daughter cells. GMCs in an embryo overexpressing Cyclin E, or in an embryo mutant for archipelago, also undergo self-renewing asymmetric divisions. Although the GMC self-renewal is independent of inscuteable and numb, the fate of the differentiating daughter is inscuteable and numb-dependent. Our results reveal that regulation of Cyclin E levels, and asymmetric distribution of Cyclin E and other determinants, confer self-renewing asymmetric division potential to precursor cells, and thus define a pathway that regulates such divisions. These results add to our understanding of maintenance and loss of pluripotential stem cell identity.     

Analysis of partner of inscuteable, a novel player of Drosophila asymmetric divisions, reveals two distinct steps in inscuteable apical localization

Asymmetric localization is a prerequisite for inscuteable (insc) to function in coordinating and mediating asymmetric cell divisions in Drosophila. We show here that Partner of Inscuteable (Pins), a new component of asymmetric divisions, is required for Inscuteable to asymmetrically localize. In the absence of pins, Inscuteable becomes cytoplasmic and asymmetric divisions of neuroblasts and mitotic domain 9 cells show defects reminiscent of insc mutants. Pins colocalizes with Insc and interacts with the region necessary and sufficient for directing its asymmetric localization. Analyses of pins function in neuroblasts reveal two distinct steps for Insc apical cortical localization: A pins-independent, bazooka-dependent initiation step during delamination (interphase) and a later maintenance step during which Baz, Pins, and Insc localization are interdependent.     

Asymmetric Numb distribution is critical for asymmetric cell division of mouse cerebral cortical stem cells and neuroblasts

Stem cells and neuroblasts derived from mouse embryos undergo repeated asymmetric cell divisions, generating neural lineage trees similar to those of invertebrates. In Drosophila, unequal distribution of Numb protein during mitosis produces asymmetric cell divisions and consequently diverse neural cell fates. We investigated whether a mouse homologue m-numb had a similar role during mouse cortical development. Progenitor cells isolated from the embryonic mouse cortex were followed as they underwent their next cell division in vitro. Numb distribution was predominantly asymmetric during asymmetric cell divisions yielding a beta-tubulin III(-) progenitor and a beta-tubulin III(+) neuronal cell (P/N divisions) and predominantly symmetric during divisions producing two neurons (N/N divisions). Cells from the numb knockout mouse underwent significantly fewer asymmetric P/N divisions compared to wild type, indicating a causal role for Numb. When progenitor cells derived from early (E10) cortex undergo P/N divisions, both daughters express the progenitor marker Nestin, indicating their immature state, and Numb segregates into the P or N daughter with similar frequency. In contrast, when progenitor cells derived from later E13 cortex (during active neurogenesis in vivo) undergo P/N divisions they produce a Nestin(+) progenitor and a Nestin(-) neuronal daughter, and Numb segregates preferentially into the neuronal daughter. Thus during mouse cortical neurogenesis, as in Drosophila neurogenesis, asymmetric segregation of Numb could inhibit Notch activity in one daughter to induce neuronal differentiation. At terminal divisions generating two neurons, Numb was symmetrically distributed in approximately 80% of pairs and asymmetrically in 20%. We found a significant association between Numb distribution and morphology: most sisters of neuron pairs with symmetric Numb were similar and most with asymmetric Numb were different. Developing cortical neurons with Numb had longer processes than those without. Numb is expressed by neuroblasts and stem cells and can be asymmetrically segregated by both. These data indicate Numb has an important role in generating asymmetric cell divisions and diverse cell fates during mouse cortical development.     

Tre1 GPCR signaling orients stem cell divisions in the Drosophila central nervous system

During development, directional cell division is a major mechanism for establishing the orientation of tissue growth. Drosophila neuroblasts undergo asymmetric divisions perpendicular to the overlying epithelium to produce descendant neurons on the opposite side, thereby orienting initial neural tissue growth. However, the mechanism remains elusive. We provide genetic evidence that extrinsic GPCR signaling determines the orientation of cortical polarity underlying asymmetric divisions of neuroblasts relative to the epithelium. The GPCR Tre1 activates the G protein oα subunit in neuroblasts by interacting with the epithelium to recruit Pins, which regulates spindle orientation. Because Pins associates with the Par-complex via Inscuteable, Tre1 consequently recruits the polarity complex to orthogonally orient the polarity axis to the epithelium. Given the universal role of the Par complex in cellular polarization, we propose that the GPCR-Pins system is a comprehensive mechanism controlling tissue polarity by orienting polarized stem cells and their divisions.     

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.     

Extrinsic cues orient the cell division axis in Drosophila embryonic neuroblasts

Cell polarity must be integrated with tissue polarity for proper development. The Drosophila embryonic central nervous system (CNS) is a highly polarized tissue; neuroblasts occupy the most apical layer of cells within the CNS, and lie just basal to the neural epithelium. Neuroblasts are the CNS progenitor cells and undergo multiple rounds of asymmetric cell division, ;budding off' smaller daughter cells (GMCs) from the side opposite the epithelium, thereby positioning neuronal/glial progeny towards the embryo interior. It is unknown whether this highly stereotypical orientation of neuroblast divisions is controlled by an intrinsic cue (e.g. cortical mark) or an extrinsic cue (e.g. cell-cell signal). Using live imaging and in vitro culture, we find that neuroblasts in contact with epithelial cells always ;bud off' GMCs in the same direction, opposite from the epithelia-neuroblast contact site, identical to what is observed in vivo. By contrast, isolated neuroblasts 'bud off' GMCs at random positions. Imaging of centrosome/spindle dynamics and cortical polarity shows that in neuroblasts contacting epithelial cells, centrosomes remained anchored and cortical polarity proteins localize at the same epithelia-neuroblast contact site over subsequent cell cycles. In isolated neuroblasts, centrosomes drifted between cell cycles and cortical polarity proteins showed a delay in polarization and random positioning. We conclude that embryonic neuroblasts require an extrinsic signal from the overlying epithelium to anchor the centrosome/centrosome pair at the site of epithelial-neuroblast contact and for proper temporal and spatial localization of cortical Par proteins. This ensures the proper coordination between neuroblast cell polarity and CNS tissue polarity.     

Cell cycle independent role of Cyclin E during neural cell fate specification in Drosophila is mediated by its regulation of Prospero function

During development, neural progenitor cells or neuroblasts generate a great intra- and inter-segmental diversity of neuronal and glial cell types in the nervous system. In thoracic segments of the embryonic central nervous system of Drosophila, the neuroblast NB6-4t undergoes an asymmetric first division to generate a neuronal and a glial sublineage, while abdominal NB6-4a divides once symmetrically to generate only 2 glial cells. We had earlier reported a critical function for the G1 cyclin, CyclinE (CycE) in regulating asymmetric cell division in NB6-4t. Here we show that (i) this function of CycE is independent of its role in cell cycle regulation and (ii) the two functions are mediated by distinct domains at the protein level. Results presented here also suggest that CycE inhibits the function of Prospero and facilitates its cortical localization, which is critical for inducing stem cell behaviour, i.e. asymmetric cell division of NB6-4t. Furthermore our data imply that CycE is required for the maintenance of stem cell identity of most other neuroblasts.     

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.     

Direct association of Bazooka/PAR-3 with the lipid phosphatase PTEN reveals a link between the PAR/aPKC complex and phosphoinositide signaling

Cell polarity in Drosophila epithelia, oocytes and neuroblasts is controlled by the evolutionarily conserved PAR/aPKC complex, which consists of the serine-threonine protein kinase aPKC and the PDZ-domain proteins Bazooka (Baz) and PAR-6. The PAR/aPKC complex is required for the separation of apical and basolateral plasma membrane domains, for the asymmetric localization of cell fate determinants and for the proper orientation of the mitotic spindle. How the complex exerts these different functions is not known. We show that the lipid phosphatase PTEN directly binds to Baz in vitro and in vivo, and colocalizes with Baz in the apical cortex of epithelia and neuroblasts. PTEN is an important regulator of phosphoinositide turnover that antagonizes the activity of PI3-kinase. We show that Pten mutant ovaries and embryos lacking maternal and zygotic Pten function display phenotypes consistent with a function for PTEN in the organization of the actin cytoskeleton. In freshly laid eggs, the germ plasm determinants oskar mRNA and Vasa are not localized properly to the posterior cytocortex and pole cells do not form. In addition, the actin-dependent posterior movement of nuclei during early cleavage divisions does not occur and the synchrony of nuclear divisions at syncytial blastoderm stages is lost. Pten mutant embryos also show severe defects during cellularization. Our data provide evidence for a link between the PAR/aPKC complex, the actin cytoskeleton and PI3-kinase signaling mediated by PTEN.     

A protein complex containing Inscuteable and the Galpha-binding protein Pins orients asymmetric cell divisions in Drosophila

BACKGROUND: In the fruit fly Drosophila, the Inscuteable protein localises to the apical cell cortex in neuroblasts and directs both the apical-basal orientation of the mitotic spindle and the basal localisation of the protein determinants Numb and Prospero during mitosis. Asymmetric localisation of Inscuteable is initiated during neuroblast delamination by direct binding to Bazooka, an apically localised protein that contains protein-interaction motifs known as PDZ domains. How apically localised Inscuteable directs asymmetric cell divisions is unclear. RESULTS: A novel 70 kDa protein called Partner of Inscuteable (Pins) and a heterotrimeric G-protein alpha subunit were found to bind specifically to the functional domain of Inscuteable in vivo. The predicted sequence of Pins contained tetratrico-peptide repeats (TPRs) and motifs implicated in binding Galpha proteins. Pins colocalised with Inscuteable at the apical cell cortex in interphase and mitotic neuroblasts. Asymmetric localisation of Pins required both Inscuteable and Bazooka. In epithelial cells, which do not express inscuteable, Pins was not apically localised but could be recruited to the apical cortex by ectopic expression of Inscuteable. In pins mutants, these epithelial cells were not affected, but neuroblasts showed defects in the orientation of their mitotic spindle and the basal asymmetric localisation of Numb and Miranda during metaphase. Although localisation of Inscuteable in pins mutants was initiated correctly during neuroblast delamination, Inscuteable became homogeneously distributed in the cytoplasm during mitosis. CONCLUSIONS: Pins and Inscuteable are dependent on each other for asymmetric localisation in delaminated neuroblasts. The binding of Pins to Galpha protein offers the intriguing possibility that Inscuteable and Pins might orient asymmetric cell divisions by localising or locally modulating a heterotrimeric G-protein signalling cascade at the apical cell cortex.     

Drosophila APC2 and APC1 have overlapping roles in the larval brain despite their distinct intracellular localizations

The tumor suppressor APC and its homologs, first identified for a role in colon cancer, negatively regulate Wnt signaling in both oncogenesis and normal development, and play Wnt-independent roles in cytoskeletal regulation. Both Drosophila and mammals have two APC family members. We further explored the functions of the Drosophila APCs using the larval brain as a model. We found that both proteins are expressed in the brain. APC2 has a highly dynamic, asymmetric localization through the larval neuroblast cell cycle relative to known mediators of embryonic neuroblast asymmetric divisions. Adherens junction proteins also are asymmetrically localized in neuroblasts. In addition they accumulate with APC2 and APC1 in nerves formed by axons of the progeny of each neuroblast-ganglion mother cell cluster. APC2 and APC1 localize to very different places when expressed in the larval brain: APC2 localizes to the cell cortex and APC1 to centrosomes and microtubules. Despite this, they play redundant roles in the brain; while each single mutant is normal, the zygotic double mutant has severely reduced numbers of larval neuroblasts. Our experiments suggest that this does not result from misregulation of Wg signaling, and thus may involve the cytoskeletal or adhesive roles of APC proteins.     

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.     

Differential functions of G protein and Baz-aPKC signaling pathways in Drosophila neuroblast asymmetric division

Drosophila melanogaster neuroblasts (NBs) undergo asymmetric divisions during which cell-fate determinants localize asymmetrically, mitotic spindles orient along the apical-basal axis, and unequal-sized daughter cells appear. We identified here the first Drosophila mutant in the Ggamma1 subunit of heterotrimeric G protein, which produces Ggamma1 lacking its membrane anchor site and exhibits phenotypes identical to those of Gbeta13F, including abnormal spindle asymmetry and spindle orientation in NB divisions. This mutant fails to bind Gbeta13F to the membrane, indicating an essential role of cortical Ggamma1-Gbeta13F signaling in asymmetric divisions. In Ggamma1 and Gbeta13F mutant NBs, Pins-Galphai, which normally localize in the apical cortex, no longer distribute asymmetrically. However, the other apical components, Bazooka-atypical PKC-Par6-Inscuteable, still remain polarized and responsible for asymmetric Miranda localization, suggesting their dominant role in localizing cell-fate determinants. Further analysis of Gbetagamma and other mutants indicates a predominant role of Partner of Inscuteable-Galphai in spindle orientation. We thus suggest that the two apical signaling pathways have overlapping but different roles in asymmetric NB division.     

DmPAR-6 directs epithelial polarity and asymmetric cell division of neuroblasts in Drosophila

The Drosophila protein Bazooka is required for both apical-basal polarity in epithelial cells and directing asymmetric cell division in neuroblasts. Here we show that the PDZ-domain protein DmPAR-6 cooperates with Bazooka for both of these functions. DmPAR-6 colocalizes with Bazooka at the apical cell cortex of epithelial cells and neuroblasts, and binds to Bazooka in vitro. DmPAR-6 localization requires Bazooka, and mislocalization of Bazooka through overexpression redirects DmPAR-6 to ectopic sites of the cell cortex. In the absence of DmPAR-6, Bazooka fails to localize apically in neuroblasts and epithelial cells, and is distributed in the cytoplasm instead. Epithelial cells lose their apical-basal polarity in DmPAR-6 mutants, asymmetric cell divisions in neuroblasts are misorientated, and the proteins Numb and Miranda do not segregate correctly into the basal daughter cell. Bazooka and DmPAR-6 are Drosophila homologues of proteins that direct asymmetric cell division in early Caenorhabditis elegans embryos, and our results indicate that homologous protein machineries may direct this process in worms and flies.     

Patterns of cell division and expression of asymmetric cell fate determinants in postembryonic neuroblast lineages of Drosophila

We have studied the division of postembryonic neuroblasts (Nbs) in the outer proliferation center (OPC) and central brain anlagen of Drosophila. We focused our attention on three aspects of these processes: the pattern of cellular division, the topological orientation of those divisions, and the expression of asymmetric cell fate determinants. Although larval Nbs are of embryonic origin, our results indicate that their properties appear to be modified during development. Several conclusions can be summarized: (i) In early larvae, Nbs divide symmetrically to give rise to two Nbs while in the late larval brain most Nbs divide asymmetrically to bud off an intermediate ganglion mother cell (GMC) that very rapidly divides into two ganglion cells (GC). (ii) Symmetric and asymmetric divisions of OPC Nbs show tangential and radial orientations, respectively. (iii) This change in the pattern of division correlates with the expression of inscuteable, which is apically localized only in asymmetric divisions. (iv) The spindle of asymmetrically dividing Nb is always oriented on an apical-basal axis. (v) Prospero does not colocalize with Miranda in the cortical crescent of mitotic Nbs. (vi) Prospero is transiently expressed in one of the two sibling GCs generated by the division of GMCs. The implications of these results on cell fate specification and differentiation of adult brain neurons are discussed.     

The C. elegans MELK ortholog PIG-1 regulates cell size asymmetry and daughter cell fate in asymmetric neuroblast divisions

In the nematode Caenorhabditis elegans, neurons are generated from asymmetric divisions in which a mother cell divides to produce daughters that differ in fate. Here, we demonstrate that the gene pig-1 regulates the asymmetric divisions of neuroblasts that divide to produce an apoptotic cell and either a neural precursor or a neuron. In pig-1 mutants, these neuroblasts divide to produce daughters that are more equal in size, and their apoptotic daughters are transformed into their sisters, leading to the production of extra neurons. PIG-1 is orthologous to MELK, a conserved member of the polarity-regulating PAR-1/Kin1/SAD-1 family of serine/threonine kinases. Although MELK has been implicated in regulating the cell cycle, our data suggest that PIG-1, like other PAR-1 family members, regulates cell polarity.     

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.     

Drosophila Pins-binding protein Mud regulates spindle-polarity coupling and centrosome organization

The orientation of the mitotic spindle relative to the cell axis determines whether polarized cells undergo symmetric or asymmetric divisions. Drosophila epithelial cells and neuroblasts provide an ideal pair of cells to study the regulatory mechanisms involved. Epithelial cells divide symmetrically, perpendicular to the apical-basal axis. In the asymmetric divisions of neuroblasts, by contrast, the spindle reorients parallel to that axis, leading to the unequal distribution of cell-fate determinants to one daughter cell. Receptor-independent G-protein signalling involving the GoLoco protein Pins is essential for spindle orientation in both cell types. Here, we identify Mushroom body defect (Mud) as a downstream effector in this pathway. Mud directly associates and colocalizes with Pins at the cell cortex overlying the spindle pole(s) in both neuroblasts and epithelial cells. The cortical Mud protein is essential for proper spindle orientation in the two different division modes. Moreover, Mud localizes to centrosomes during mitosis independently of Pins to regulate centrosomal organization. We propose that Drosophila Mud, vertebrate NuMA and Caenorhabditis elegans Lin-5 (refs 5, 6) have conserved roles in the mechanism by which G-proteins regulate the mitotic spindle.