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.     

Asymmetric cell division

Asymmetric cell division is a conserved mechanism for partitioning information during mitosis. Over the past several years, significant progress has been made in our understanding of how cells establish polarity during asymmetric cell division and how determinants, in the form of localized proteins and mRNAs, are segregated. In particular, genetic studies in Drosophila and Caenorhabditis elegans have linked cell polarity, G protein signaling and regulation of the cytoskeleton to coordination of mitotic spindle orientation and localization of determinants. Also, several new studies have furthered our understanding of how asymmetrically localized cell fate determinants, such as the Numb, a negative regulator Notch signaling, functions in biasing cell fates in the developing nervous system in Drosophila. In vertebrates, analysis of dividing neural progenitor cells by in vivo imaging has raised questions about the role of asymmetric cell divisions during neurogenesis.     

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.     

discordia mutations specifically misorient asymmetric cell divisions during development of the maize leaf epidermis.

In plant cells, cytokinesis depends on a cytoskeletal structure called a phragmoplast, which directs the formation of a new cell wall between daughter nuclei after mitosis. The orientation of cell division depends on guidance of the phragmoplast during cytokinesis to a cortical site marked throughout prophase by another cytoskeletal structure called a preprophase band. Asymmetrically dividing cells become polarized and form asymmetric preprophase bands prior to mitosis; phragmoplasts are subsequently guided to these asymmetric cortical sites to form daughter cells of different shapes and/or sizes. Here we describe two new recessive mutations, discordia1 (dcd1) and discordia2 (dcd2), which disrupt the spatial regulation of cytokinesis during asymmetric cell divisions. Both mutations disrupt four classes of asymmetric cell divisions during the development of the maize leaf epidermis, without affecting the symmetric divisions through which most epidermal cells arise. The effects of dcd mutations on asymmetric cell division can be mimicked by cytochalasin D treatment, and divisions affected by dcd1 are hypersensitive to the effects of cytochalasin D. Analysis of actin and microtubule organization in these mutants showed no effect of either mutation on cell polarity, or on formation and localization of preprophase bands and spindles. In mutant cells, phragmoplasts in asymmetrically dividing cells are structurally normal and are initiated in the correct location, but often fail to move to the position formerly occupied by the preprophase band. We propose that dcd mutations disrupt an actin-dependent process necessary for the guidance of phragmoplasts during cytokinesis in asymmetrically dividing cells.     

Mitotic spindle orientation distinguishes stem cell and terminal modes of neuron production in the early spinal cord

Despite great insight into the molecular mechanisms that specify neuronal cell type in the spinal cord, cell behaviour underlying neuron production in this tissue is largely unknown. In other neuroepithelia, divisions with a perpendicular cleavage plane at the apical surface generate symmetrical cell fates, whereas a parallel cleavage plane generates asymmetric daughters, a neuron and a progenitor in a stem cell mode, and has been linked to the acquisition of neuron-generating ability. Using a novel long-term imaging assay, we have monitored single cells in chick spinal cord as they transit mitosis and daughter cells become neurons or divide again. We reveal new morphologies accompanying neuron birth and show that neurons are generated concurrently by asymmetric and terminal symmetric divisions. Strikingly, divisions that generate two progenitors or a progenitor and a neuron both exhibit a wide range of cleavage plane orientations and only divisions that produce two neurons have an exclusively perpendicular orientation. Neuron-generating progenitors are also distinguished by lengthening cell cycle times, a finding supported by cell cycle acceleration on exposure to fibroblast growth factor (FGF), an inhibitor of neuronal differentiation. This study provides a novel, dynamic view of spinal cord neurogenesis and supports a model in which cleavage plane orientation/mitotic spindle position does not assign neuron-generating ability, but functions subsequent to this step to distinguish stem cell and terminal modes of neuron production.     

Neurogenesis and asymmetric cell division

The astonishing cellular diversity in the central nervous system (CNS) arises from neural progenitors which can undergo different modes of symmetric and asymmetric divisions to self-renew as well as produce differentiated neuronal and glial progeny. Drosophila CNS neural progenitor cells, neuroblasts, have been utilised as a model to stimulate the understanding of the processes of asymmetric division, generation of neuronal lineages and, more recently, stem cell biology in vertebrates. Here we review some recent developments involving Drosophila and mammalian neural progenitor cells, highlighting some similarities and differences in the mechanisms that regulate their divisions during neurogenesis.     

Mammalian neurogenesis requires Treacle-Plk1 for precise control of spindle orientation, mitotic progression, and maintenance of neural progenitor cells

The cerebral cortex is a specialized region of the brain that processes cognitive, motor, somatosensory, auditory, and visual functions. Its characteristic architecture and size is dependent upon the number of neurons generated during embryogenesis and has been postulated to be governed by symmetric versus asymmetric cell divisions, which mediate the balance between progenitor cell maintenance and neuron differentiation, respectively. The mechanistic importance of spindle orientation remains controversial, hence there is considerable interest in understanding how neural progenitor cell mitosis is controlled during neurogenesis. We discovered that Treacle, which is encoded by the Tcof1 gene, is a novel centrosome- and kinetochore-associated protein that is critical for spindle fidelity and mitotic progression. Tcof1/Treacle loss-of-function disrupts spindle orientation and cell cycle progression, which perturbs the maintenance, proliferation, and localization of neural progenitors during cortical neurogenesis. Consistent with this, Tcof1(+/-) mice exhibit reduced brain size as a consequence of defects in neural progenitor maintenance. We determined that Treacle elicits its effect via a direct interaction with Polo-like kinase1 (Plk1), and furthermore we discovered novel in vivo roles for Plk1 in governing mitotic progression and spindle orientation in the developing mammalian cortex. Increased asymmetric cell division, however, did not promote increased neuronal differentiation. Collectively our research has therefore identified Treacle and Plk1 as novel in vivo regulators of spindle fidelity, mitotic progression, and proliferation in the maintenance and localization of neural progenitor cells. Together, Treacle and Plk1 are critically required for proper cortical neurogenesis, which has important implications in the regulation of mammalian brain size and the pathogenesis of congenital neurodevelopmental disorders such as microcephaly.     

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.     

Cancer stem cell, niche and EGFR decide tumor development and treatment response: A bio-computational simulation study

Recent research in cancer biology has suggested the hypothesis that tumors are initiated and driven by a small group of cancer stem cells (CSCs). Furthermore, cancer stem cell niches have been found to be essential in determining fates of CSCs, and several signaling pathways have been proven to play a crucial role in cellular behavior, which could be two important factors in cancer development. To better understand the progression, heterogeneity and treatment response of breast cancer, especially in the context of CSCs, we propose a mathematical model based on the cell compartment method. In this model, three compartments of cellular subpopulations are constructed: CSCs, progenitor cells (PCs), and terminal differentiated cells (TCs). Moreover, (1) the cancer stem cell niche is, considered by modeling its effect on division patterns (symmetric or asymmetric) of CSCs, and (2) the EGFR signaling pathway is integrated by modeling its role in cell proliferation, apoptosis. Our simulation results indicate that (1) a higher probability for symmetric division of CSC may result in a faster expansion of tumor population, and for a larger number of niches, the tumor grows at a slower rate, but the final tumor volume is larger; (2) higher EGFR expression correlates to tumors with larger volumes while a saturation function is observed, and (3) treatments that inhibit tyrosine kinase activity of EGFR may not only repress the tumor volume, but also decrease the CSCs percentages by shifting CSCs from symmetric divisions to asymmetric divisions. These findings suggest that therapies should be designed to effectively control or eliminate the symmetric division of CSCs and to reduce or destroy the CSC niches.     

When timing is everything: role of cell cycle regulation in asymmetric division

Asymmetric division is a fundamental mechanism of generating cell diversity during development. One of its hallmarks is asymmetric localization during mitosis of proteins that specify daughter cell fate. Studies in Drosophila show that subcellular localization of many proteins required for asymmetric division of neuronal progenitors correlates with progression through mitosis. Yet, how cell cycle and asymmetric division machineries cooperate remains unclear. Recent data show that (1) key cell cycle regulators are required for asymmetric localization of cell fate determinants and for cell fate determination and (2) molecules that mediate asymmetric division can also act to modulate proliferation potential of progenitor cells.     

Cell biology of stem cells: an enigma of asymmetry and self-renewal

Stem cells present a vast, new terrain of cell biology. A central question in stem cell research is how stem cells achieve asymmetric divisions to replicate themselves while producing differentiated daughter cells. This hallmark of stem cells is manifested either strictly during each mitosis or loosely among several divisions. Current research has revealed the crucial roles of niche signaling, intrinsic cell polarity, subcellular localization mechanism, asymmetric centrosomes and spindles, as well as cell cycle regulators in establishing self-renewing asymmetry during stem cell division. Much of this progress has benefited from studies in model stem cell systems such as Drosophila melanogaster neuroblasts and germline stem cells and mammalian skin stem cells. Further investigations of these questions in diverse types of stem cells will significantly advance our knowledge of cell biology and allow us to effectively harness stem cells for therapeutic applications.     

Morphological asymmetry in dividing retinal progenitor cells

For the understanding of histogenetic events in the 3-D retinal neuroepithelium, direct observation of the progenitor cells and their morphological changes is required. A slice culture method has been developed by which the behavior of single progenitor cells can be monitored. Although it has been believed that each retinal progenitor cell loses its basal process while it is in M phase, it is reported here that the process is retained throughout M phase and is inherited by one daughter cell, which can be a neuron or a progenitor cell. Daughter neurons used an inherited process for neuronal translocation and positioning. In divisions that produced two mitotic daughters, both of which subsequently divided to form four granddaughter cells, only one daughter cell inherited the original basal process while the other extended a new process. Interestingly, behavioral differences were often noted between such mitotic sisters in the trajectory of interkinetic nuclear movement, cell cycle length, and the composition of the granddaughter pair. Therefore, "symmetric" (progenitor --> progenitor + progenitor) divisions are in fact morphologically asymmetric, and the behavior of the mitotic daughters can often be asymmetric, indicating the necessity for studying possible associations between the process inheritance and the cell fate choice.     

Cell polarity and asymmetric division in the peripheral nervous system of Drosophila

During metazoan development, cell fate diversity is generated in part by asymmetric cell divisions, in which mother cells divide to produce two daughter cells with distinct developmental potentials. Adoption of different cell fates often relies on the polarised distribution and unequal segregation of cell-fate determinants. Unequal segregation of cell-fate determinants requires that the mother cell becomes polarised prior to mitosis. In response to this polarisation, cell-fate determinants localise asymmetrically and the mitotic spindle lines up with the pole to which cell-fate determinants accumulate, thereby leading to their unequal partitioning upon cytokinesis. I review here the regulatory mechanisms that establish cell asymmetry and orient this asymmetry relative to the body axis in the sensory organ lineages of Drosophila.     

Mammalian inscuteable regulates spindle orientation and cell fate in the developing retina

During mammalian neurogenesis, progenitor cells can divide with the mitotic spindle oriented parallel or perpendicular to the surface of the neuroepithelium. Perpendicular divisions are more likely to be asymmetric and generate one progenitor and one neuronal precursor. Whether the orientation of the mitotic spindle actually determines their asymmetric outcome is unclear. Here, we characterize a mammalian homolog of Inscuteable (mInsc), a key regulator of spindle orientation in Drosophila. mInsc is expressed temporally and spatially in a manner that suggests a role in orienting the mitotic spindle in the developing nervous system. Using retroviral RNAi in rat retinal explants, we show that downregulation of mInsc inhibits vertical divisions. This results in enhanced proliferation, consistent with a higher frequency of symmetric divisions generating two proliferating cells. Our results suggest that the orientation of neural progenitor divisions is important for cell fate specification in the retina and determines their symmetric or asymmetric outcome.     

Simulating Temporal Organization of Histogenesis

The paper discusses a discrete model of hepatic plate histogenesis based on the dissymmetry distribution times of cell cycles. The model is based on the assumption of the finality of cell time as an internal parameter of a single cycle of biological spacetime. Another assumption is the divisibility of maternal cell time in half between daughter cells, which makes it possible to consider the stem cell macrocycle in histogenesis as a system of nested cell cycles with progressively decreasing time. In addition, it was expected that at each step of cell division in homogeneous histogenesis, a asymmetric distribution of reference of cell time management by mitosis, which is related to the intron region of the genome. The obtained numerical model agrees with the characteristics of the architectonics of the liver, the limited number of cell divisions, and the frequency distribution of mitosis and aging as the extinction process of cells with short cycles.     

Heterogenic Final Cell Cycle by Chicken Retinal Lim1 Horizontal Progenitor Cells Leads to Heteroploid Cells with a Remaining Replicated Genome

Retinal progenitor cells undergo apical mitoses during the process of interkinetic nuclear migration and newly generated post-mitotic neurons migrate to their prospective retinal layer. Whereas this is valid for most types of retinal neurons, chicken horizontal cells are generated by delayed non-apical mitoses from dedicated progenitors. The regulation of such final cell cycle is not well understood and we have studied how Lim1 expressing horizontal progenitor cells (HPCs) exit the cell cycle. We have used markers for S- and G2/M-phase in combination with markers for cell cycle regulators Rb1, cyclin B1, cdc25C and p27Kip1 to characterise the final cell cycle of HPCs. The results show that Lim1+ HPCs are heterogenic with regards to when and during what phase they leave the final cell cycle. Not all horizontal cells were generated by a non-apical (basal) mitosis; instead, the HPCs exhibited three different behaviours during the final cell cycle. Thirty-five percent of the Lim1+ horizontal cells was estimated to be generated by non-apical mitoses. The other horizontal cells were either generated by an interkinetic nuclear migration with an apical mitosis or by a cell cycle with an S-phase that was not followed by any mitosis. Such cells remain with replicated DNA and may be regarded as somatic heteroploids. The observed heterogeneity of the final cell cycle was also seen in the expression of Rb1, cyclin B1, cdc25C and p27Kip1. Phosphorylated Rb1-Ser608 was restricted to the Lim1+ cells that entered S-phase while cyclin B1 and cdc25C were exclusively expressed in HPCs having a basal mitosis. Only HPCs that leave the cell cycle after an apical mitosis expressed p27Kip1. We speculate that the cell cycle heterogeneity with formation of heteroploid cells may present a cellular context that contributes to the suggested propensity of these cells to generate cancer when the retinoblastoma gene is mutated.     

Male gametogenesis without centrioles

The orientation of the mitotic spindle plays a central role in specifying stem cell-renewal by enabling interaction of the daughter cells with external cues: the daughter cell closest to the hub region is instructed to self-renew, whereas the distal one starts to differentiate. Here, we have analyzed male gametogenesis in DSas-4 Drosophila mutants and we have reported that spindle alignment and asymmetric divisions are properly executed in male germline stem cells that lack centrioles. Spermatogonial divisions also correctly proceed in the absence of centrioles, giving rise to cysts of 16 primary spermatocytes. By contrast, abnormal meiotic spindles assemble in primary spermatocytes. These results point to different requirements for centrioles during male gametogenesis of Drosophila. Spindle formation during germ cell mitosis may be successfully supported by an acentrosomal pathway that is inadequate to warrant the proper execution of meiosis.     

KIF18B is a cell type–specific regulator of spindle orientation in the epidermis

Proper spindle orientation is required for asymmetric cell division and the establishment of complex tissue architecture. In the developing epidermis, spindle orientation requires a conserved cortical protein complex of LGN/NuMA/dynein-dynactin. However, how microtubule dynamics are regulated to interact with this machinery and properly position the mitotic spindle is not fully understood. Furthermore, our understanding of the processes that link spindle orientation during asymmetric cell division to cell fate specification in distinct tissue contexts remains incomplete. We report a role for the microtubule catastrophe factor KIF18B in regulating microtubule dynamics to promote spindle orientation in keratinocytes. During mitosis, KIF18B accumulates at the cell cortex, colocalizing with the conserved spindle orientation machinery. In vivo we find that KIF18B is required for oriented cell divisions within the hair placode, the first stage of hair follicle morphogenesis, but is not essential in the interfollicular epidermis. Disrupting spindle orientation in the placode, using mutations in either KIF18B or NuMA, results in aberrant cell fate marker expression of hair follicle progenitor cells. These data functionally link spindle orientation to cell fate decisions during hair follicle morphogenesis. Taken together, our data demonstrate a role for regulated microtubule dynamics in spindle orientation in epidermal cells. This work also highlights the importance of spindle orientation during asymmetric cell division to dictate cell fate specification.     

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.