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.     

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.     

Numb inhibits membrane localization of Sanpodo,a four-pass transmembrane protein,to promote asymmetric divisions in Drosophila

Cellular diversity is a fundamental characteristic of complex organisms, and the Drosophila CNS has proved an informative paradigm for understanding the mechanisms that create cellular diversity. One such mechanism is the asymmetric localization of Numb to ensure that sibling cells respond differently to the extrinsic Notch signal and, thus, adopt distinct fates (A and B). Here we focus on the only genes known to function specifically to regulate Notch-dependent asymmetric divisions: sanpodo and numb. We demonstrate that sanpodo, which specifies the Notch-dependent fate (A), encodes a four-pass transmembrane protein that localizes to the cell membrane in the A cell and physically interacts with the Notch receptor. We also show that Numb, which inhibits Notch signaling to specify the default fate (B), physically associates with Sanpodo and inhibits Sanpodo membrane localization in the B cell. Our findings suggest a model in which Numb inhibits Notch signaling through the regulation of Sanpodo membrane localization.     

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.     

Ethanolamine kinase controls neuroblast divisions in Drosophila mushroom bodies

The Drosophila mushroom bodies (MBs), paired brain structures composed of vertical and medial lobes, achieve their final organization at metamorphosis. The alpha lobe absent (ala) mutant randomly lacks either the vertical lobes or two of the median lobes. We characterize the ala axonal phenotype at the single-cell level, and show that the ala mutation affects Drosophila ethanolamine (Etn) kinase activity and induces Etn accumulation. Etn kinase is overexpressed in almost all cancer cells. We demonstrate that this enzymatic activity is required in MB neuroblasts to allow a rapid rate of cell division at metamorphosis, linking Etn kinase activity with mitotic progression. Tight control of the pace of neuroblast division is therefore crucial for completion of the developmental program in the adult brain.     

Distinct roles of Galphai and Gbeta13F subunits of the heterotrimeric G protein complex in the mediation of Drosophila neuroblast asymmetric divisions

The asymmetric division of Drosophila neuroblasts involves the basal localization of cell fate determinants and the generation of an asymmetric, apicobasally oriented mitotic spindle that leads to the formation of two daughter cells of unequal size. These features are thought to be controlled by an apically localized protein complex comprising of two signaling pathways: Bazooka/Drosophila atypical PKC/Inscuteable/DmPar6 and Partner of inscuteable (Pins)/Galphai; in addition, Gbeta13F is also required. However, the role of Galphai and the hierarchical relationship between the G protein subunits and apical components are not well defined. Here we describe the isolation of Galphai mutants and show that Galphai and Gbeta13F play distinct roles. Galphai is required for Pins to localize to the cortex, and the effects of loss of Galphai or pins are highly similar, supporting the idea that Pins/Galphai act together to mediate various aspects of neuroblast asymmetric division. In contrast, Gbeta13F appears to regulate the asymmetric localization/stability of all apical components, and Gbeta13F loss of function exhibits phenotypes resembling those seen when both apical pathways have been compromised, suggesting that it acts upstream of the apical pathways. Importantly, our results have also revealed a novel aspect of apical complex function, that is, the two apical pathways act redundantly to suppress the formation of basal astral microtubules in neuroblasts.     

Signalling molecules,growth regulators and cell cycle control in Drosophila

During the development of a given organ or tissue within a multicellular organism, growth and patterning are controlled in a coordinated manner by the activity of a discrete number of signalling molecules and their corresponding pathways to give rise to a well formed structure with a particular size, shape and pattern. Understanding how cells of different tissues or organs translate in a context dependent manner the activity of these pathways into an activation or repression of the cell cycle machinery is one of the most intriguing questions in developmental and cancer biology nowadays. Here we revise the different roles of the signalling molecules Notch and Wingless in the regulation of cell cycle progression in the developing eye and wing imaginal discs of Drosophila and propose that depending on how growth regulators are regulated in a context dependent manner by the activity of these pathways, signalling molecules might have tumour suppressor or oncogene activity.     

Differential effects of Drosophila mastermind on asymmetric cell fate specification and neuroblast formation

During neurogenesis in the ventral nerve cord of the Drosophila embryo, Notch signaling participates in the pathway that mediates asymmetric fate specification to daughters of secondary neuronal precursor cells. In the NB4-2 --> GMC-1 --> RP2/sib lineage, a well-studied neuronal lineage in the ventral nerve cord, Notch signaling specifies sib fate to one of the daughter cells of GMC-1. Notch mediates this process via Mastermind (Mam). Loss of function for mam, similar to loss of function for Notch, results in GMC-1 symmetrically dividing to generate two RP2 neurons. Loss of function for mam also results in a severe neurogenic phenotype. In this study, we have undertaken a functional analysis of the Mam protein. We show that while ectopic expression of a truncated Mam protein induces a dominant-negative neurogenic phenotype, it has no effect on asymmetric fate specification. This truncated Mam protein rescues the loss of asymmetric specification phenotype in mam in an allele-specific manner. We also show an interallelic complementation of loss-of-asymmetry defect. Our results suggest that Mam proteins might associate during the asymmetric specification of cell fates and that the N-terminal region of the protein plays a role in this process.     

Apical complex genes control mitotic spindle geometry and relative size of daughter cells in Drosophila neuroblast and pI asymmetric divisions

Drosophila neuroblast asymmetric divisions generate two daughters of unequal size and fate. A complex of apically localized molecules mediates basal localization of cell fate determinants and apicobasal orientation of the mitotic spindle, but how daughter cell size is controlled remains unclear. Here we show that mitotic spindle geometry and unequal daughter cell size are controlled by two parallel pathways (Bazooka/DaPKC and Pins/G alpha i) within the apical complex. While the localized activity of either pathway alone is sufficient to mediate the generation of an asymmetric mitotic spindle and unequal size neuroblast daughters, loss of both pathways results in symmetric divisions. In sensory organ precursors, Bazooka/DaPKC and Pins/G alpha i localize to opposite sides of the cortex and function in opposition to generate a symmetric spindle.     

Cortical development and asymmetric cell divisions

The development of the mammalian neocortex involves rounds of symmetric and asymmetric cell division of neural progenitors to fulfill needs of both self-renewal of progenitors and production of differe...     

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.     

Drosophila Anillin is unequally required during asymmetric cell divisions of the PNS

During Drosophila embryogenesis, timely and orderly asymmetric cell divisions ensure the correct number of each cell type that make up the sensory organs of the larval PNS. We report a role of scraps, Drosophila Anillin, during these divisions. Anillin, a constitutive member of the contractile ring is essential for cytokinesis in Drosophila and vertebrates. During embryogenesis we find that zygotically transcribed scraps is required specifically for the unequal cell divisions, those in which cytokinesis occurs in an “off-centred” manner, of the pIIb and pIIIb neuronal precursor cells, but not the equal cell divisions of the lineage related precursor cells. Complementation and genetic rescue studies demonstrate this effect results from zygotic scraps and leads to polyploidy, ectopic mitosis, and loss of the neuronal precursor daughter cells. The net result of which is the formation of incomplete sense organs and embryonic lethality.     

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.     

Two types of asymmetric divisions in the Drosophila sensory organ precursor cell lineage

Asymmetric partitioning of cell-fate determinants during development requires coordinating the positioning of these determinants with orientation of the mitotic spindle. In the Drosophila peripheral nervous system, sensory organ progenitor cells (SOPs) undergo several rounds of division to produce five cells that give rise to a complete sensory organ. Here we have observed the asymmetric divisions that give rise to these cells in the developing pupae using green fluorescent protein fusion proteins. We find that spindle orientation and determinant localization are tightly coordinated at each division. Furthermore, we find that two types of asymmetric divisions exist within the sensory organ precursor cell lineage: the anterior-posterior pI cell-type division, where the spindle remains symmetric throughout mitosis, and the strikingly neuroblast-like apical-basal division of the pIIb cell, where the spindle exhibits a strong asymmetry at anaphase. In both these divisions, the spindle reorientates to position itself perpendicular to the region of the cortex containing the determinant. On the basis of these observations, we propose that two distinct mechanisms for controlling asymmetric cell divisions occur within the same lineage in the developing peripheral nervous system in Drosophila.     

C. elegans HAM-1 positions the cleavage plane and regulates apoptosis in asymmetric neuroblast divisions

Asymmetric cell division occurs when a mother cell divides to generate two distinct daughter cells, a process that promotes the generation of cellular diversity in metazoans. During Caenorhabditis elegans development, the asymmetric divisions of neural progenitors generate neurons, neural support cells and apoptotic cells. C. elegans HAM-1 is an asymmetrically distributed cortical protein that regulates several of these asymmetric neuroblast divisions. Here, we show that HAM-1 is a novel protein and define residues important for HAM-1 function and distribution to the cell cortex. Our phenotypic analysis of ham-1 mutant embryos suggests that HAM-1 controls only neuroblast divisions that produce apoptotic cells. Moreover, ham-1 mutant embryos contain many unusually large cell-death corpses. An investigation of this corpse phenotype revealed that it results from a reversal of neuroblast polarity. A misplacement of the neuroblast cleavage plane generates daughter cells of abnormal size, with the apoptotic daughters larger than normal. Thus, HAM-1 regulates the position of the cleavage plane, apoptosis and mitotic potential in C. elegans asymmetric cell divisions.     

The GC kinase Fray and Mo25 regulate Drosophila asymmetric divisions

Drosophila neuroblasts provide an excellent model for asymmetric cell divisions, where cell-fate determinants such as Miranda localize at the basal cortex and segregate to one daughter cell. Mechanisms underlying this process, however, remain elusive. We found that Mo25 and the GC kinase Fray act in this regulation. mo25 and fray mutants show an indistinguishable defect in Miranda localization. On the other hand, Drosophila Mo25 interacts with the tumor suppressor kinase Lkb1 in vivo, as have shown in mammals. Overexpression of Lkb1, which accumulates in the cell cortex, drastically relocalizes both Mo25 and Fray from the cytoplasm to the cortex, causing the same phenotype as mo25-mutant neuroblasts. Recovery from this defect caused by Lkb1 overexpression requires simultaneous overexpression of Mo25 and Fray. We suggest from those results that Mo25 and Fray operate together or in the same pathway in Drosophila asymmetric processes, and that their function counterbalances Lkb1.     

Hypoxia and nitric oxide induce a rapid, reversible cell cycle arrest of the Drosophila syncytial divisions

Cells can respond to reductions in oxygen (hypoxia) by metabolic adaptations, quiescence or cell death. The nuclear division cycles of syncytial stage Drosophila melanogaster embryos reversibly arrest upon hypoxia. We examined this rapid arrest in real time using a fusion of green fluorescent protein and histone 2A. In addition to an interphase arrest, mitosis was specifically blocked in metaphase, much like a checkpoint arrest. Nitric oxide, recently proposed as a hypoxia signal in Drosophila, induced a reversible arrest of the nuclear divisions comparable with that induced by hypoxia. Syncytial stage embryos die during prolonged hypoxia, whereas post-gastrulation embryos (cellularized) survive. We examined ATP levels and morphology of syncytial and cellularized embryos arrested by hypoxia, nitric oxide, or cyanide. Upon oxygen deprivation, the ATP levels declined only slightly in cellularized embryos and more substantially in syncytial embryos. Reversal of hypoxia restored ATP levels and relieved the cell cycle and developmental arrests. However, morphological abnormalities suggested that syncytial embryos suffered irreversible disruption of developmental programs. Our results suggest that nitric oxide plays a role in the response of the syncytial embryo to hypoxia but that it is not the sole mediator of these responses.