Drosophila E-cadherin regulates the orientation of asymmetric cell division in the sensory organ lineage

BACKGROUND: Generation of cell-fate diversity in Metazoan depends in part on asymmetric cell divisions in which cell-fate determinants are asymmetrically distributed in the mother cell and unequally partitioned between daughter cells. The polarization of the mother cell is a prerequisite to the unequal segregation of cell-fate determinants. In the Drosophila bristle lineage, two distinct mechanisms are known to define the axis of polarity of the pI and pIIb cells. Frizzled (Fz) signaling regulates the planar orientation of the pI division, while Inscuteable (Insc) directs the apical-basal polarity of the pIIb cell. The orientation of the asymmetric division of the pIIa cell is identical to the one of its mother cell, the pI cell, but, in contrast, is regulated by an unknown Insc- and Fz-independent mechanism. RESULTS: DE-Cadherin-Catenin complexes are shown to localize at the cell contact between the two cells born from the asymmetric division of the pI cell. The mitotic spindle of the dividing pIIa cell rotates to line up with asymmetrically localized DE-Cadherin-Catenin complexes. While a complete loss of DE-Cadherin function disrupts the apical-basal polarity of the epithelium, both a partial loss of DE-Cadherin function and expression of a dominant-negative form of DE-Cadherin affect the orientation of the pIIa division. Furthermore, expression of dominant-negative DE-Cadherin also affects the position of Partner of Inscuteable (Pins) and Bazooka, two asymmetrically localized proteins known to regulate cell polarity. These results show that asymmetrically distributed Cad regulates the orientation of asymmetric cell division. CONCLUSIONS: We describe a novel mechanism involving a specialized Cad-containing cortical region by which a daughter cell divides with the same orientation as its mother cell.     

Localization-dependent and -independent roles of numb contribute to cell-fate specification in Drosophila

During asymmetric cell division, protein determinants are segregated into one of the two daughter cells. The Numb protein acts as a segregating determinant during both mouse and Drosophila development. In flies, Numb localizes asymmetrically and is required for cell-fate specification in the central and peripheral nervous systems, as well as during muscle and heart development. Whether its asymmetric segregation is important to the performance of these functions is not firmly established. Here, we demonstrate that Numb acts both in a localization-dependent and in a localization-independent manner. We have generated numb mutants that affect only the asymmetric localization of the protein during mitosis. We demonstrate that asymmetric segregation of Numb into one of the two daughter cells is absolutely essential for cell-fate specification in the Drosophila peripheral nervous system. Numb localization is also essential in MP2 neuroblasts in the central nervous system and during muscle development. Surprisingly, in dividing ganglion mother cells or during heart development, Numb function is independent of its ability to segregate asymmetrically in mitosis. Our results suggest that two classes of asymmetric cell division exist, each with different requirements for asymmetric inheritance of cell-fate determinants.     

Frizzled regulates localization of cell-fate determinants and mitotic spindle rotation during asymmetric cell division

Cell-fate diversity is generated in part by the unequal segregation of cell-fate determinants during asymmetric cell divisions. In the Drosophila pupa, the pI sense organ precursor cell is polarized along the anterior-posterior axis of the fly and divides asymmetrically to generate a posterior pIIa cell and an anterior pIIb cell. The anterior pIIb cell specifically inherits the determinant Numb and the adaptor protein Partner of Numb (Pon). By labelling both the Pon crescent and the microtubules in living pupae, we show that determinants localize at the anterior cortex before mitotic-spindle formation, and that the spindle forms with random orientation and rotates to line up with the Pon crescent. By imaging living frizzled (fz) mutant pupae we show that Fz regulates the orientation of the polarity axis of pI, the initiation of spindle rotation and the unequal partitioning of determinants. We conclude that Fz participates in establishing the polarity of pI.     

The planar cell polarity protein Strabismus promotes Pins anterior localization during asymmetric division of sensory organ precursor cells in Drosophila

Cell fate diversity is generated in part by the unequal segregation of cell-fate determinants during asymmetric cell division. In the Drosophila bristle lineage, the sensory organ precursor (pI) cell is polarized along the anteroposterior (AP) axis by Frizzled (Fz) receptor signaling. We show here that Fz localizes at the posterior apical cortex of the pI cell prior to mitosis, whereas Strabismus (Stbm) and Prickle (Pk), which are also required for AP polarization of the pI cell, co-localize at the anterior apical cortex. Thus, asymmetric localization of Fz, Stbm and Pk define two opposite cortical domains prior to mitosis of the pI cell. At mitosis, Stbm forms an anterior crescent that overlaps with the distribution of Partner of Inscuteable (Pins) and Discs-large (Dlg), two components of the anterior Dlg-Pins-Galphai complex that regulates the localization of cell-fate determinants. At prophase, Stbm promotes the anterior localization of Pins. By contrast, Dishevelled (Dsh) acts antagonistically to Stbm by excluding Pins from the posterior cortex. We propose that the Stbm-dependent recruitment of Pins at the anterior cortex of the pI cell is a novel read-out of planar cell polarity.     

The orientation of cell division influences cell-fate choice in the developing mammalian retina

Asymmetric segregation of cell-fate determinants during cell division plays an important part in generating cell diversity in invertebrates. We showed previously that cells in the neonatal rat retina divide at various orientations and that some dividing cells asymmetrically distribute the cell-fate determinant Numb to the two daughter cells. Here, we test the possibility that such asymmetric divisions contribute to retinal cell diversification. We have used long-term videomicroscopy of green-fluorescent-protein (GFP)-labeled retinal explants from neonatal rats to visualize the plane of cell division and follow the differentiation of the daughter cells. We found that cells that divided with a horizontal mitotic spindle, where both daughter cells should inherit Numb, tended to produce daughters that became the same cell type, whereas cells that divided with a vertical mitotic spindle, where only one daughter cell should inherit Numb, tended to produce daughters that became different. Moreover, overexpression of Numb in the dividing cells promoted the development of photoreceptor cells at the expense of interneurons and Müller glial cells. These findings indicate that the plane of cell division influences cell-fate choice in the neonatal rat retina and support the hypothesis that the asymmetric segregation of Numb normally influences some of these choices.     

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.     

A mutation in the ATP2 gene abrogates the age asymmetry between mother and daughter cells of the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae reproduces by asymmetric cell division, or budding. In each cell division, the daughter cell is usually smaller and younger than the mother cell, as defined by the number of divisions it can potentially complete before it dies. Although individual yeast cells have a limited life span, this age asymmetry between mother and daughter ensures that the yeast strain remains immortal. To understand the mechanisms underlying age asymmetry, we have isolated temperature-sensitive mutants that have limited growth capacity. One of these clonal-senescence mutants was in ATP2, the gene encoding the beta-subunit of mitochondrial F(1), F(0)-ATPase. A point mutation in this gene caused a valine-to-isoleucine substitution at the ninetieth amino acid of the mature polypeptide. This mutation did not affect the growth rate on a nonfermentable carbon source. Life-span determinations following temperature shift-down showed that the clonal-senescence phenotype results from a loss of age asymmetry at 36 degrees, such that daughters are born old. It was characterized by a loss of mitochondrial membrane potential followed by the lack of proper segregation of active mitochondria to daughter cells. This was associated with a change in mitochondrial morphology and distribution in the mother cell and ultimately resulted in the generation of cells totally lacking mitochondria. The results indicate that segregation of active mitochondria to daughter cells is important for maintenance of age asymmetry and raise the possibility that mitochondrial dysfunction may be a normal cause of aging. The finding that dysfunctional mitochondria accumulated in yeasts as they aged and the propensity for old mother cells to produce daughters depleted of active mitochondria lend support to this notion. We propose, more generally, that age asymmetry depends on partition of active and undamaged cellular components to the progeny and that this "filter" breaks down with age.     

Stem cell ageing and non-random chromosome segregation

Adult stem cells maintain the mature tissues of metazoans. They do so by reproducing in such a way that their progeny either differentiate, and thus contribute functionally to a tissue, or remain uncommitted and replenish the stem cell pool. Because ageing manifests as a general decline in tissue function, diminished stem cell-mediated tissue maintenance may contribute to age-related pathologies. Accordingly, the mechanisms by which stem cell regenerative potential is sustained, and the extent to which these mechanisms fail with age, are fundamental determinants of tissue ageing. Here, we explore the mechanisms of asymmetric division that account for the sustained fitness of adult stem cells and the tissues that comprise them. In particular, we summarize the theory and experimental evidence underlying non-random chromosome segregation-a mitotic asymmetry arising from the unequal partitioning of chromosomes according to the age of their template DNA strands. Additionally, we consider the possible consequences of non-random chromosome segregation, especially as they relate to both replicative and chronological ageing in stem cells. While biased segregation of chromosomes may sustain stem cell replicative potential by compartmentalizing the errors derived from DNA synthesis, it might also contribute to the accrual of replication-independent DNA damage in stem cells and thus hasten chronological ageing.     

Endocytosis by Numb breaks Notch symmetry at cytokinesis

Cell-fate diversity can be generated by the unequal segregation of the Notch regulator Numb at mitosis in both vertebrates and invertebrates. Whereas the mechanisms underlying unequal inheritance of Numb are understood, how Numb antagonizes Notch has remained unsolved. Live imaging of Notch in sensory organ precursor cells revealed that nuclear Notch is detected at cytokinesis in the daughter cell that does not inherit Numb. Numb and Sanpodo act together to regulate Notch trafficking and establish directional Notch signalling at cytokinesis. We propose that unequal segregation of Numb results in increased endocytosis in one daughter cell, hence asymmetry of Notch at the cytokinetic furrow, directional signalling and binary fate choice.     

Dare to be different: asymmetric cell division in Drosophila, C. elegans and vertebrates

One widespread mechanism for the generation of diverse cell types is the unequal inheritance of cell fate determinants. Several such determinants have been identified in the fruitfly Drosophila melanogaster and the worm Caenorhabditis elegans and the molecular machinery responsible for their asymmetric segregation is beginning to be unraveled. To divide asymmetrically, cells establish an axis of polarity, orient the mitotic spindle along this axis and localize cell fate determinants to one side of the cell. During cytokinesis, determinants are then segregated into one of the two daughter cells where they direct cell fate. Here, we outline the steps and factors that are involved in this process in Drosophila and C. elegans and discuss their potential conservation in vertebrates.     

Asymmetric distribution of mitochondria and of spindle microtubules in opposite directions in differential mitosis of germ line cells in Acricotopus

Additional chromosomes present only in the germ line are a specific feature of the Orthocladiinae, a subfamily of the Chironomidae. During the complex chromosome cycle in the orthocladiid Acricotopus lucidus, about half of the germ-line-limited chromosomes (Ks) are eliminated in the first division of the primary germ cells. Following normal gonial mitoses, the reduction in the number of Ks is compensated for, in the last mitosis prior to meiosis, by a monopolar movement of the unseparated Ks, while the somatic chromosomes (Ss) segregate equally. This differential mitosis produces daughter cells with different chromosome constitutions and diverse developmental fates. A preferential segregation of mitochondria occurs to one pole associated with an asymmetric formation of the mitotic spindle. This has been detected in living gonial cells in both sexes by using MitoTracker probes and fluorochrome-labelled paclitaxel (taxol). In males, the resulting unequal partitioning of mitochondria to the daughter cells is equalised by the transport of mitochondria through a permanent cytoplasmic bridge from the aberrant spermatocyte to the primary spermatocyte. This asymmetry in the distribution and in the segregation of cytoplasmic components in differential gonial mitosis in Acricotopus may be involved in the process of cell-fate determination. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Asymmetric division and cosegregation of template DNA strands in adult muscle satellite cells

Satellite cells assure postnatal skeletal muscle growth and repair. Despite extensive studies, their stem cell character remains largely undefined. Using pulse-chase labelling with BrdU to mark the putative stem cell niche, we identify a subpopulation of label-retaining satellite cells during growth and after injury. Strikingly, some of these cells display selective template-DNA strand segregation during mitosis in the muscle fibre in vivo, as well as in culture independent of their niche, indicating that genomic DNA strands are nonequivalent. Furthermore, we demonstrate that the asymmetric cell-fate determinant Numb segregates selectively to one daughter cell during mitosis and before differentiation, suggesting that Numb is associated with self-renewal. Finally, we show that template DNA cosegregates with Numb in label-retaining cells that express the self-renewal marker Pax7. The cosegregation of 'immortal' template DNA strands and their link with the asymmetry apparatus has important implications for stem cell biology and cancer.     

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.     

The Wnt receptor Ryk controls specification of GABAergic neurons versus oligodendrocytes during telencephalon development

GABAergic neurons and oligodendrocytes originate from progenitors within the ventral telencephalon. However, the molecular mechanisms that control neuron-glial cell-fate segregation, especially how extrinsic factors regulate cell-fate changes, are poorly understood. We have discovered that the Wnt receptor Ryk promotes GABAergic neuron production while repressing oligodendrocyte formation in the ventral telencephalon. We demonstrate that Ryk controls the cell-fate switch by negatively regulating expression of the intrinsic oligodendrogenic factor Olig2 while inducing expression of the interneuron fate determinant Dlx2. In addition, we demonstrate that Ryk is required for GABAergic neuron induction and oligodendrogenesis inhibition caused by Wnt3a stimulation. Furthermore, we showed that the cleaved intracellular domain of Ryk is sufficient to regulate the cell-fate switch by regulating the expression of intrinsic cell-fate determinants. These results identify Ryk as a multi-functional receptor that is able to transduce extrinsic cues into progenitor cells, promote GABAergic neuron formation, and inhibit oligodendrogenesis during ventral embryonic brain development.     

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.     

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.     

Drosophila mechanoreceptors as a model for studying asymmetric cell division

Asymmetric cell division (ACD) is one of the processes creating the overall diversity of cell types in multicellular organisms. The essence of this process is that the daughter cells exit from it being different from both the parental cell and one another in their ability to further differentiation and specialization. The large bristles (macrochaetae) that are regularly arranged on the surface of the Drosophila adult function as mechanoreceptors, and since their development requires ACD, they have been extensively used as a model system for studying the genetic control of this process. Each macrochaete is composed of four specialized cells, the progeny resulting from several ACDs from a single sensory organ precursor (SOP) cell, which differentiates from the ectodermal cells of the wing imaginal disc in the third-instar larva and pupa. In this paper we review the experimental data on the genes and their products controlling the ACDs of the SOP cell and its daughter cells, and their further specialization. We discuss the main mechanisms determining the time when the cell enters ACD, as well as the mechanisms providing for the structural characteristics of asymmetric division, namely, polar distribution of protein determinants (Numb and Neuralized), orientation of the division spindle relative to these determinants, and unequal segregation of the determinants specifying the direction of daughter cell development.     

Brief cytochalasin-induced disruption of microfilaments during a critical interval in 1-cell C. elegans embryos alters the partitioning of developmental instructions to the 2-cell embryo

We are investigating the involvement of the microfilament cytoskeleton in the development of early Caenorhabditis elegans embryos. We previously reported that several cytoplasmic movements in the zygote require that the microfilament cytoskeleton remain intact during a narrow time interval approximately three-quarters of the way through the first cell cycle. In this study, we analyze the developmental consequences of brief, cytochalasin D-induced microfilament disruption during the 1-cell stage. Our results indicate that during the first cell cycle microfilaments are important only during the critical time interval for the 2-cell embryo to undergo the correct pattern of subsequent divisions and to initiate the differentiation of at least 4 tissue types. Disruption of microfilaments during the critical interval results in aberrant division and P-granule segregation patterns, generating some embryos that we classify as 'reverse polarity', 'anterior duplication', and 'posterior duplication' embryos. These altered patterns suggest that microfilament disruption during the critical interval leads to the incorrect distribution of developmental instructions responsible for early pattern formation. The strict correlation between unequal division, unequal germ-granule partitioning, and the generation of daughter cells with different cell cycle periods observed in these embryos suggests that the three processes are coupled. We hypothesize that (1) an 'asymmetry determinant', normally located at the posterior end of the zygote, governs asymmetric cell division, germ-granule segregation, and the segregation of cell cycle timing elements during the first cell cycle, and (2) the integrity or placement of this asymmetry determinant is sensitive to microfilament disruption during the critical time interval.     

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.     

Rotation and asymmetry of the mitotic spindle direct asymmetric cell division in the developing central nervous system

The asymmetric segregation of cell-fate determinants and the generation of daughter cells of different sizes rely on the correct orientation and position of the mitotic spindle. In the Drosophila embryo, the determinant Prospero is localized basally and is segregated equally to daughters of similar cell size during epidermal cell division. In contrast, during neuroblast division Prospero is segregated asymmetrically to the smaller daughter cell. This simple switch between symmetric and asymmetric segregation is achieved by changing the orientation of cell division: neural cells divide in a plane perpendicular to that of epidermoblast division. Here, by labelling mitotic spindles in living Drosophila embryos, we show that neuroblast spindles are initially formed in the same axis as epidermal cells, but rotate before cell division. We find that daughter cells of different sizes arise because the spindle itself becomes asymmetric at anaphase: apical microtubules elongate, basal microtubules shorten, and the midbody moves basally until it is positioned asymmetrically between the two spindle poles. This observation contradicts the widely held hypothesis that the cleavage furrow is always placed midway between the two centrosomes.