Mechanisms of asymmetric cell division

Asymmetric cell division (ACD) is a critical mechanism to generate diverse cell types. Drosophila male germline stem cells (GSCs) divide asymmetrically by orienting their spindle toward the niche component hub cells. The hub cells secrete the self-renewal ligand Upd to activate a cytokine receptor-like Dome in GSCs. In the talk, Yamashita shows that Upd-Dome dictates spindle orientation via direct binding to Eb1, a major microtubule regulator. Dome-Eb1 interaction is mediated by SxIP Eb1-binding motif in the cytoplasmic tail of Dome. Strikingly, Yamashita discovers that cytokine-receptor-Eb1 axis is conserved in mammalian cells, where gp130, a mammalian homolog of Dome, regulates centrosome orientation at the immunological synapse in T cells via SxIP motif. Yamashita’s study reveals an evolutionarily conserved mechanism to orient centrosomes and spindles, coupled with engagement of ligand and receptor.

Along which axis chromosomes segregate is a key determinant of cell fate in asymmetrically dividing cells. In budding yeast, transition from mitosis to G1 is tightly coordinated with the position of the mitotic spindle to assure chromosome segregation along the cell polarity axis prior to mitotic exit and cytokinesis. A centrosome-associated signalling cascade, the mitotic exit network (MEN) triggers exit from mitosis, while the surveillance mechanism of the spindle position checkpoint (SPOC) inhibits MEN if the mitotic spindle fails to align along the cell polarity axis. The mother cell cortex and centrosome-associated kinase Kin4 lies in the heart of SPOC signalling. The Pereira group performed a genetic screen to identify novel regulators of Kin4 and SPOC. In the talk, the analysis of a sub-set of novel regulators will be presented. Furthermore, Pereira discusses how temporal and compartment-specific regulatory networks determine mitotic exit in SPOC and MEN signalling.

The polarised partitioning of proteins in cells underlies asymmetric cell division, which is an important driver of development and cellular diversity. Like most cells, the budding yeast Saccharomyces cerevisiae divides asymmetrically to give two distinct daughter cells. This asymmetry mimics that seen in metazoans and the key regulatory proteins are conserved from yeast to human. A well-known example of an asymmetric protein is the transcription factor Ace2, which localises specifically to the daughter nucleus, where it drives a daughter-specific transcriptional network. Thorpe performed a reverse genetic screen to look for regulators of asymmetry based on Ace2 localization phenotype. Thorpe screened a collection of essential genes in order to analyse the effect of core cellular processes in asymmetric cell division. The group identified members of the evolutionary conserved FACT chromatin-remodelling complex and show that these are required for both asymmetric and cell cycle regulated localization of Ace2.

Asymmetric cell divisions balance stem cell proliferation and differentiation to sustain tissue morphogenesis and homeostasis. During asymmetric divisions, fate determinants and niche contacts segregate unequally between daughters, but little is known on how this is achieved mechanistically. In Drosophila neuroblasts and murine mammary stem cells, the association of the spindle orientation protein LGN with the stem cell adaptor Inscuteable has been connected to asymmetry. Mapelli will present the crystal structure of LGN in complex with the asymmetric domain of Inscuteable, which reveals a stable tetrameric arrangement of intertwined molecules. Biochemical analyses show that Insc:LGN tetramers constitute stable cores for the assembly of conserved Par3-Insc-LGN-G(alpha)i^GDP complexes, which cannot be dissociated by NuMA. In mammary stem cells, the asymmetric domain of Insc suffices to drive asymmetric fate, and reverts aberrant symmetric divisions induced by p53 loss. Intriguingly, structural studies also revealed that the C-terminal portion of NuMA assembles in hetero-hexamers with LGN-TPR, which are the functional oligomeric units driving mitotic spindle orientation. Based on these findings, we propose that two independent pools of LGN contribute to asymmetry in different ways: the Insc-bound pool by coordinating the distribution of fate determinants to promote asymmetric fate specification, and the more abundant NuMA-bound pool by providing cortical information to orient the division plane in a Dynein dependent manner.

Different cell types are generated by asymmetric segregation of fate determinants during neuroblast divisions in the developing nervous system of Drosophila. The establishment of cortical polarity precedes the localization of determinants such as Miranda, but how this is controlled remains unclear. Using live cell imaging of endogenous reporters for the Par complex and Miranda, Januschke reveals the highly dynamic nature of neuroblast polarization. Januschke shows that Miranda uses two different modes to interact with the NB cortex. In interphase Miranda binds to the plasma membrane via a previously identified aPKC regulated basic and hydrophobic motif. After nuclear envelope breakdown Miranda switches to a binding mode dependent on actin and myosin activity to anchor it to a basal affinity zone. Therefore, different mechanisms control establishment of Miranda asymmetry and maintenance. His findings support a model integrating aPKC-mediated phospho-regulation of determinant localization and differential binding to the actin cortex.

One focus of regenerative medicine is to efficiently and safely replace retinal ganglion cells (RGCs), the output neurons of the retina, which are lost upon glaucoma and optic neuropathies leading to irreversible blindness. To this end, a tightly controlled expansion of the source cell (stem, progenitor or even differentiated neuronal cells) as well as its efficient and safe reprogramming into functional RGCs is needed. Achieving this requires that we understand the complex crosstalk of cell fate determinants, chromatin regulators and self-renewal factors that are at work during the normal genesis of RGCs in vivo. The bHLH transcription factor Ath5 (Atoh7) plays a crucial role in instructing RGC fate acquisition of retinal progenitor cells as well as induced pluripotent stem cells and Müller glia. Expression of ath5, however, is not decisive for RGC commitment and cells escape the RGC fate while progressing through self-renewal states. Poggi is interested in disentangling the integrated molecular interactions, from daughter cell inheritance to transcriptional regulation, which allow ath5-expressing progenitors to elude the RGC differentiation pathway upon asymmetric cell division. To understand this, Poggi began with interrogating gene regulatory networks controlled by Ath5, and to examine them in the physiological cellular context of the three-dimensionally patterned retinal tissue of the developing zebrafish embryo. Here Poggi provides insights suggesting how reciprocal feedbacks between Ath5 and factors influencing multipotency and self-renewal might intersect during asymmetric cell division, to restrict the RGC fate choice of retinal progenitor cells.

Tapering body parts are common in nature, but little is known about the developmental mechanisms by which taper arises. The simple 40 cell notochord of the invertebrate chordate Ciona forms a tapered rod, and the Veeman group are using it as a model for dissecting the mechanisms of tapering. Much of the taper reflects the cells at the front and back of the notochord being progressively smaller in volume than cells in the middle. Veeman used a genetic fate mapping strategy based on mosaic expression of an electroporated transgene to show that asymmetric division is the major driver of these anterior to posterior cell volume differences. Cells in the anterior of the notochord primordium divide to give smaller anterior daughters, and cells in the posterior of the primordium divide to give smaller posterior daughters. The volume asymmetries seen are modest compared with many cases of asymmetric division, but two consecutive rounds of division with complex patterns of asymmetry lead to important changes in cell size and ultimately help control the shape of an entire chordate organ. Veeman is currently investigating the cellular and molecular mechanisms driving these novel asymmetric divisions. The mitotic spindle becomes robustly oriented along the AP axis in these cells, but it is not clear if it is being displaced. The group are working to test an alternate hypothesis that some of these asymmetric divisions might involve a centred spindle in the context of an asymmetrically shaped cell.

During development of central nervous system (CNS) neural progenitors must be able to self-renew while producing neurons by undergoing series of asymmetric divisions. During early stages of zebrafish embryonic development, differentiated neurons appear regularly spaced along the anterior posterior axis of the spinal cord.  This suggests that a mechanism that spaces neural progenitors’ asymmetric divisions or differentiating neurons must exist in this region. To determine the cellular and molecular mechanisms that can regulate neuronal patterning in the neural tube, Alexandre used live-imaging in zebrafish embryo to monitor neural progenitors’ divisions and neurons differentiating in vivo. Alexandre discovered that neuronal committed progenitors transiently elongate two long basal processes along the antero-posterior axis of the neural tube. The Alexandre group has evidence that signals delivered by these long processes may prevent neighbours from differentiating. This is the first cellular behaviour found in a vertebrate system that can regulate neuronal patterning in the neural tube.

Stem cells are highly abundant during early development but become rare in most adult organs. Stem cell numbers must then be tightly regulated during development, but the molecular mechanisms causing stem cells to exit proliferation at a specific time are unclear. To address the mechanisms triggering stem cell exit during development Homem used Drosophila neural stem cells, the neuroblasts. Neuroblasts undergo size and fate asymmetric divisions to self-renew and generate more differentiated cells. Neuroblasts proliferate during development but all exit cell cycle and disappear before adulthood. Homem’s data show that changes in energy metabolism induced by the steroid hormone Ecdysone together with transcription regulator Mediator initiate an irreversible cascade of events leading to neuroblast differentiation. An increase in the levels of oxidative phosphorylation in neuroblasts leads to uncoupling between cell cycle from cell growth. This results in progressive reduction in neuroblast cell size and ultimately in terminal differentiation. Homem’s findings show that neuroblast size control can be modified by systemic hormonal signalling and reveal a unique connection between metabolism and proliferation in stem cells.

Unequal maturation of centrosomes has been associated with differential fate choices in several models of asymmetric cell division, including vertebrate neural stem cells. Nevertheless, signalling molecules taking advantage of the intrinsic centrosome asymmetry to instruct binary fate choices in sister cells have yet to be identified. Morin’s group found that the mono-ubiquitin ligase Mindbomb1 (Mib1), a regulator of the Notch pathway, localizes to centriolar satellites associated with the daughter centrosome of chick spinal cord progenitors. Using live imaging and fate tracking in the neural tube, we show that during asymmetric divisions, the centrosome carrying this pool of Mib1 is inherited by the prospective neuron. However, in symmetric divisions, a second pool of Mib1 associated with the Golgi apparatus in interphase is released during mitosis, aggregates on the free centrosome, and compensates for the initial Mib1 centrosomal asymmetry. Remarkably, as development of the neural tube proceeds, Mib1 is progressively lost from the Golgi apparatus, correlating with the reduction in symmetric proliferative divisions. Thus, the Golgi apparatus may represent a storage compartment compensating for the centrosome asymmetry of Mib1 in proliferative divisions. As neurogenesis progresses, this compensatory mechanism fades out and neurogenic divisions become predominant. Finally, Morin shows that preventing Mib1 centrosomal association in dividing cells hinders Notch signalling and delays neuronal differentiation in daughter cells. Thus, Morin establishes for the first time a link between centrosome asymmetry and Notch signalling, and propose that changes in the subcellular localization of Mib1 in neural progenitors are instrumental for the progression of neurogenesis.

During brain development, neural stem cells define specific niches within different cortical layers to control their cellular environment. Generally, communication to other cells or surrounding matrix by biochemical signalling pathways such as protein-protein interactions or soluble factors have been well studied. Much less studied, however, is how physical properties of the niche can influence behaviour, growth, and differentiation of cells. Among physical properties, stiffness of the matrix has been shown to have direct effects on fate determination in certain stem cell types in vitro. While some studies indicate that stiffness may influence the fate of cultured neural stem cells by unknown mechanisms, little evidence exists on whether this principle holds true during the physiological development of the mammalian brain in vivo. Here, Kosodo has characterized tissue stiffness of the developing mouse brain cortex by using atomic force microscopy to seek a link to the cell fate determination of neural cells via mechanosignalling pathways, and found specific spatiotemporal shifts in stiffness during brain formation. The research aims to establish a novel concept for mammalian neurogenesis and brain development, and the outcomes will likely influence the fields of stem cell and developmental biology by clarifying unknown mechanosignalling mechanisms of somatic stem cells.