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.     

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.     

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.     

Twins/PP2A regulates aPKC to control neuroblast cell polarity and self-renewal

Asymmetric cell division is a mechanism for generating cell diversity as well as maintaining stem cell homeostasis in both Drosophila and mammals. In Drosophila, larval neuroblasts are stem cell-like progenitors that divide asymmetrically to generate neurons of the adult brain. Mitotic neuroblasts localize atypical protein kinase C (aPKC) to their apical cortex. Cortical aPKC excludes cortical localization of Miranda and its cargo proteins Prospero and Brain tumor, resulting in their partitioning into the differentiating, smaller ganglion mother cell (GMC) where they are required for neuronal differentiation. In addition to aPKC, the kinases Aurora-A and Polo also regulate neuroblast self-renewal, but the phosphatases involved in neuroblast self-renewal have not been identified. Here we report that aPKC is in a protein complex in vivo with Twins, a Drosophila B-type protein phosphatase 2A (PP2A) subunit, and that Twins and the catalytic subunit of PP2A, called Microtubule star (Mts), are detected in larval neuroblasts. Both Twins and Mts are required to exclude aPKC from the basal neuroblast cortex: twins mutant brains, twins mutant single neuroblast mutant clones, or mts dominant negative single neuroblast clones all show ectopic basal cortical localization of aPKC. Consistent with ectopic basal aPKC is the appearance of supernumerary neuroblasts in twins mutant brains or twins mutant clones. We conclude that Twins/PP2A is required to maintain aPKC at the apical cortex of mitotic neuroblasts, keeping it out of the differentiating GMC, and thereby maintaining neuroblast homeostasis.     

Drosophila homologs of mammalian TNF/TNFR-related molecules regulate segregation of Miranda/Prospero in neuroblasts

During neuroblast (NB) divisions, cell fate determinants Prospero (Pros) and Numb, together with their adaptor proteins Miranda (Mira) and Partner of Numb, localize to the basal cell cortex at metaphase and segregate exclusively to the future ganglion mother cells (GMCs) at telophase. In inscuteable mutant NBs, these basal proteins are mislocalized during metaphase. However, during anaphase/telophase, these mutant NBs can partially correct these earlier localization defects and redistribute cell fate determinants as crescents to the region where the future GMC "buds" off. This compensatory mechanism has been referred to as "telophase rescue". We demonstrate that the Drosophila homolog of the mammalian tumor-necrosis factor (TNF) receptor-associated factor (DTRAF1) and Eiger (Egr), the homolog of the mammalian TNF, are required for telophase rescue of Mira/Pros. DTRAF1 localizes as an apical crescent in metaphase NBs and this apical localization requires Bazooka (Baz) and Egr. The Mira/Pros telophase rescue seen in inscuteable mutant NBs requires DTRAF1. Our data suggest that DTRAF1 binds to Baz and acts downstream of Egr in the Mira/Pros telophase rescue pathway.     

Asymmetric Segregation of the Double-Stranded RNA Binding Protein Staufen2 during Mammalian Neural Stem Cell Divisions Promotes Lineage Progression

Asymmetric cell divisions are a fundamental feature of neural development, and misregulation can lead to brain abnormalities or tumor formation. During an asymmetric cell division, molecular determinants are segregated preferentially into one daughter cell to specify its fate. An important goal is to identify the asymmetric determinants in neural progenitor cells, which could be tumor suppressors or inducers of specific neural fates. Here, we show that the double-stranded RNA-binding protein Stau2 is distributed asymmetrically during progenitor divisions in the developing mouse cortex, preferentially segregating into the Tbr2(+) neuroblast daughter, taking with it a subset of RNAs. Knockdown of Stau2 stimulates differentiation and overexpression produces periventricular neuronal masses, demonstrating its functional importance for normal cortical development. We immunoprecipitated Stau2 to examine its cargo mRNAs, and found enrichment for known asymmetric and basal cell determinants, such as Trim32, and identified candidates, including a subset involved in primary cilium function.     

HLH-14 is a C. elegans achaete-scute protein that promotes neurogenesis through asymmetric cell division

Achaete-Scute basic helix-loop-helix (bHLH) proteins promote neurogenesis during metazoan development. In this study, we characterize a C. elegans Achaete-Scute homolog, HLH-14. We find that a number of neuroblasts express HLH-14 in the C. elegans embryo, including the PVQ/HSN/PHB neuroblast, a cell that generates the PVQ interneuron, the HSN motoneuron and the PHB sensory neuron. hlh-14 mutants lack all three of these neurons. The fact that HLH-14 promotes all three classes of neuron indicates that C. elegans proneural bHLH factors may act less specifically than their fly and mammalian homologs. Furthermore, neural loss in hlh-14 mutants results from a defect in an asymmetric cell division: the PVQ/HSN/PHB neuroblast inappropriately assumes characteristics of its sister cell, the hyp7/T blast cell. We argue that bHLH proteins, which control various aspects of metazoan development, can control cell fate choices in C. elegans by regulating asymmetric cell divisions. Finally, a reduction in the function of hlh-2, which encodes the C. elegans E/Daughterless bHLH homolog, results in similar neuron loss as hlh-14 mutants and enhances the effects of partially reducing hlh-14 function. We propose that HLH-14 and HLH-2 act together to specify neuroblast lineages and promote neuronal fate.     

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.     

The Drosophila myosin VI Jaguar is required for basal protein targeting and correct spindle orientation in mitotic neuroblasts

Asymmetric cell divisions generate cellular diversity. In Drosophila, embryonic neuroblasts target cell fate determinants basally, rotate their spindles by 90 degrees to align with the apical-basal axis, and divide asymmetrically in a stem cell-like fashion. In this process, apically localized Bazooka recruits Inscuteable and other proteins to form an apical complex, which then specifies spindle orientation and basal localization of the cell fate determinants and their adapter proteins such as Miranda. Here we report that Miranda localization requires the unconventional myosin VI Jaguar (Jar). In jar null mutant embryos, Miranda is delocalized and the spindle is misoriented, but the Inscuteable crescent remains apical. Miranda directly binds to Jar, raising the possibility that Miranda and its associated proteins are translocated basally by this actin-based motor. Our studies demonstrate that a class VI myosin is necessary for basal protein targeting and spindle orientation in neuroblasts.     

Heterotrimeric G proteins regulate daughter cell size asymmetry in Drosophila neuroblast divisions

Cell division often generates unequally sized daughter cells by off-center cleavages, which are due to either displacement of mitotic spindles or their asymmetry. Drosophila neuroblasts predominantly use the latter mechanism to divide into a large apical neuroblast and a small basal ganglion mother cell (GMC), where the neural fate determinants segregate. Apically localized components regulate both the spindle asymmetry and the localization of the determinants. Here, we show that asymmetric spindle formation depends on signaling mediated by the G beta subunit of heterotrimeric G proteins. G beta 13F distributes throughout the neuroblast cortex. Its lack induces a large symmetric spindle and causes division into nearly equal-sized cells with normal segregation of the determinants. In contrast, elevated G beta 13F activity generates a small spindle, suggesting that this factor suppresses spindle development. Depletion of the apical components also results in the formation of a small symmetric spindle at metaphase. Therefore, the apical components and G beta 13F affect the mitotic spindle shape oppositely. We propose that differential activation of G beta signaling biases spindle development within neuroblasts and thereby causes asymmetric spindles. Furthermore, the multiple equal cleavages of G beta mutant neuroblasts accompany neural defects; this finding suggests indispensable roles of eccentric division in assuring the stem cell properties of 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.     

Miranda couples oskar mRNA/Staufen complexes to the bicoid mRNA localization pathway

The double-stranded RNA binding protein Staufen is required for the microtubule-dependent localization of bicoid and oskar mRNAs to opposite poles of the Drosophila oocyte and also mediates the actin-dependent localization of prospero mRNA during the asymmetric neuroblast divisions. The posterior localization of oskar mRNA requires Staufen RNA binding domain 2, whereas prospero mRNA localization mediated the binding of Miranda to RNA binding domain 5, suggesting that different Staufen domains couple mRNAs to distinct localization pathways. Here, we show that the expression of Miranda during mid-oogenesis targets Staufen/oskar mRNA complexes to the anterior of the oocyte, resulting in bicaudal embryos that develop an abdomen and pole cells instead of the head and thorax. Anterior Miranda localization requires microtubules, rather than actin, and depends on the function of Exuperantia and Swallow, indicating that Miranda links Staufen/oskar mRNA complexes to the bicoid mRNA localization pathway. Since Miranda is expressed in late oocytes and bicoid mRNA localization requires the Miranda-binding domain of Staufen, Miranda may play a redundant role in the final step of bicoid mRNA localization. Our results demonstrate that different Staufen-interacting proteins couple Staufen/mRNA complexes to distinct localization pathways and reveal that Miranda mediates both actin- and microtubule-dependent mRNA localization.     

Drosophila nonmuscle myosin II promotes the asymmetric segregation of cell fate determinants by cortical exclusion rather than active transport

Cell fate diversity can be achieved through the asymmetric segregation of cell fate determinants. In the Drosophila embryo, neuroblasts divide asymmetrically and in a stem cell fashion. The determinants Prospero and Numb localize in a basal crescent and are partitioned from neuroblasts to their daughters (GMCs). Here we show that nonmuscle myosin II regulates asymmetric cell division by an unexpected mechanism, excluding determinants from the apical cortex. Myosin II is activated by Rho kinase and restricted to the apical cortex by the tumor suppressor Lethal (2) giant larvae. During prophase and metaphase, myosin II prevents determinants from localizing apically. At anaphase and telophase, myosin II moves to the cleavage furrow and appears to "push" rather than carry determinants into the GMC. Therefore, the movement of myosin II to the contractile ring not only initiates cytokinesis but also completes the partitioning of cell fate determinants from the neuroblast to its daughter.     

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.     

A primary cell culture of Drosophila postembryonic larval neuroblasts to study cell cycle and asymmetric division

Drosophila melanogaster is a key model system that has greatly contributed to the advance of developmental biology through its extensive and sophisticated genetics. Nevertheless, only a few in vitro approaches are available in Drosophila to complement genetic studies in order to better elucidate developmental mechanisms at the cellular and molecular level. Here we present a dissociated cell culture system generated from the optic lobes of Drosophila larval brain. This culture system makes it feasible to study the proliferative properties of Drosophila postembryonic Nbs by allowing BrdU pulse and chase assays, as well as detailed immunocytochemical analysis with molecular markers. These immunofluorescence experiments allowed us to conclude that localization of asymmetric cell division markers such as Inscuteable, Miranda, Prospero and Numb is cell autonomous. By time-lapse video recording we have observed interesting cellular features of postembryonic neurogenesis such us the polarized genesis of the neuroblast progeny, the extremely short ganglion mother cell (GMC) cell cycle, and the last division of a neuroblast lineage. The combination of this cell culture system and genetic tools of Drosophila will provide a powerful experimental model for the analysis of cell cycle and asymmetric cell division of neural progenitor cells.     

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.     

Brat is a Miranda cargo protein that promotes neuronal differentiation and inhibits neuroblast self-renewal

An important question in stem cell biology is how a cell decides to self-renew or differentiate. Drosophila neuroblasts divide asymmetrically to self-renew and generate differentiating progeny called GMCs. Here, we report that the Brain tumor (Brat) translation repressor is partitioned into GMCs via direct interaction with the Miranda scaffolding protein. In brat mutants, another Miranda cargo protein (Prospero) is not partitioned into GMCs, GMCs fail to downregulate neuroblast gene expression, and there is a massive increase in neuroblast numbers. Single neuroblast clones lacking Prospero have a similar phenotype. We conclude that Brat suppresses neuroblast stem cell self-renewal and promotes neuronal differentiation.     

Uncovering the link between malfunctions in Drosophila neuroblast asymmetric cell division and tumorigenesis

Asymmetric cell division is a developmental process utilized by several organisms. On the most basic level, an asymmetric division produces two daughter cells, each possessing a different identity or fate. Drosophila melanogaster progenitor cells, referred to as neuroblasts, undergo asymmetric division to produce a daughter neuroblast and another cell known as a ganglion mother cell (GMC). There are several features of asymmetric division in Drosophila that make it a very complex process, and these aspects will be discussed at length. The cell fate determinants that play a role in specifying daughter cell fate, as well as the mechanisms behind setting up cortical polarity within neuroblasts, have proved to be essential to ensuring that neurogenesis occurs properly. The role that mitotic spindle orientation plays in coordinating asymmetric division, as well as how cell cycle regulators influence asymmetric division machinery, will also be addressed. Most significantly, malfunctions during asymmetric cell division have shown to be causally linked with neoplastic growth and tumor formation. Therefore, it is imperative that the developmental repercussions as a result of asymmetric cell division gone awry be understood.