Revisão Acesso aberto Revisado por pares

Molecular mechanisms of asymmetric divisions in mammary stem cells

2016; Springer Nature; Volume: 17; Issue: 12 Linguagem: Inglês

10.15252/embr.201643021

ISSN

1469-3178

Autores

Angela Santoro, Thalia Vlachou, Manuel Carminati, Pier Giuseppe Pelicci, Marina Mapelli,

Tópico(s)

Cancer Cells and Metastasis

Resumo

Review21 November 2016free access Molecular mechanisms of asymmetric divisions in mammary stem cells Angela Santoro Department of Experimental Oncology, European Institute of Oncology, Milan, Italy Search for more papers by this author Thalia Vlachou Department of Experimental Oncology, European Institute of Oncology, Milan, Italy Search for more papers by this author Manuel Carminati Department of Experimental Oncology, European Institute of Oncology, Milan, Italy Search for more papers by this author Pier Giuseppe Pelicci Corresponding Author [email protected] Department of Experimental Oncology, European Institute of Oncology, Milan, Italy Search for more papers by this author Marina Mapelli Corresponding Author [email protected] orcid.org/0000-0001-8502-0649 Department of Experimental Oncology, European Institute of Oncology, Milan, Italy Search for more papers by this author Angela Santoro Department of Experimental Oncology, European Institute of Oncology, Milan, Italy Search for more papers by this author Thalia Vlachou Department of Experimental Oncology, European Institute of Oncology, Milan, Italy Search for more papers by this author Manuel Carminati Department of Experimental Oncology, European Institute of Oncology, Milan, Italy Search for more papers by this author Pier Giuseppe Pelicci Corresponding Author [email protected] Department of Experimental Oncology, European Institute of Oncology, Milan, Italy Search for more papers by this author Marina Mapelli Corresponding Author [email protected] orcid.org/0000-0001-8502-0649 Department of Experimental Oncology, European Institute of Oncology, Milan, Italy Search for more papers by this author Author Information Angela Santoro1,‡, Thalia Vlachou1,‡, Manuel Carminati1, Pier Giuseppe Pelicci *,1 and Marina Mapelli *,1 1Department of Experimental Oncology, European Institute of Oncology, Milan, Italy ‡These authors contributed equally to this work *Corresponding author. Tel: +39 02 57489831; E-mail: [email protected] *Corresponding author. Tel: +39 02 94375018; E-mail: [email protected] EMBO Rep (2016)17:1700-1720https://doi.org/10.15252/embr.201643021 See the Glossary for abbreviations used in this article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Stem cells have the remarkable ability to undergo proliferative symmetric divisions and self-renewing asymmetric divisions. Balancing of the two modes of division sustains tissue morphogenesis and homeostasis. Asymmetric divisions of Drosophila neuroblasts (NBs) and sensory organ precursor (SOP) cells served as prototypes to learn what we consider now principles of asymmetric mitoses. They also provide initial evidence supporting the notion that aberrant symmetric divisions of stem cells could correlate with malignancy. However, transferring the molecular knowledge of circuits underlying asymmetry from flies to mammals has proven more challenging than expected. Several experimental approaches have been used to define asymmetry in mammalian systems, based on daughter cell fate, unequal partitioning of determinants and niche contacts, or proliferative potential. In this review, we aim to provide a critical evaluation of the assays used to establish the stem cell mode of division, with a particular focus on the mammary gland system. In this context, we will discuss the genetic alterations that impinge on the modality of stem cell division and their role in breast cancer development. Glossary ACD asymmetric cell division AurkA Aurora-A kinase BCC basal cell carcinoma BMP bone morphogenetic protein Cre-ER Cre recombinase CSC cancer stem cell DP dermal papilla ECM extracellular matrix EGF epidermal growth factor GMC ganglion mother cell GSC germline stem cell HFSC hair follicle stem cell HTT huntingtin ISC intestinal stem cell IF immunofluorescence MaSC mammary stem cell MRU mammary repopulating unit NB neuroblast SCC squamous cell carcinoma SCD symmetric cell division SC stem cell SFE sphere-forming efficiency SHH sonic hedgehog SOP sensory organ precursor TA transit amplifying TEB terminal end bud Introduction Stem cells are defined by their capacity of long-term self-renewal coupled with the ability to generate differentiated progeny, both of which enable them to sustain morphogenetic programs and tissue homeostasis. Self-renewal and differentiation are accomplished through a single mitosis, in which at least one of the two daughters retains stemness (asymmetric divisions; ACD). Alternatively, stem cells can undergo symmetric proliferative divisions (SCD) generating two stem cells (Fig 1A). In multicellular organisms, to prevent aberrant growth or loss of tissue, the balance between asymmetric and symmetric mitoses must be exquisitely controlled in time and space throughout the entire lifespan. The defining mechanisms governing such a balance constitute key unresolved issues in adult stem cell biology. Figure 1. Self-renewing symmetric and asymmetric divisions sustain tissue morphogenesis and homeostasis (A) Scheme of asymmetric versus symmetric self-renewing stem cell divisions. In ACD (left), self-renewal is attained by unequal partitioning of fate determinants and niche contacts, so that only one cell retains stemness (pale yellow), while the other one is committed to differentiation (gold). In SCD (right), stem cells proliferate by equally distributing cellular components between the two daughter cells, generating two stem cells. (B) Intrinsic ACDs of Drosophila neuroblasts delaminated from the neuroepithelium generating two differently sized daughters: one neuroblast and one ganglion mother cell (GMC). The larger neuroblast inherits the apical Baz/Par6/aPKC polarity complex (purple crescent), the spindle orientation proteins Pins, Mud, Gαi, and Inscuteable (cyan crescent) and maintains stemness. The smaller GMC inherits fate determinants (brown dots), which activate a neuronal differentiation program, and the mother centrosome (red circle). (C) Drosophila male GSCs divide asymmetrically producing one stem cell contacting the niche (Hub) through adherens junctions (magenta rods), and a distal daughter differentiating into a gonioblast and positioned among somatic cyst cells. The mother centrosome (red circle) segregates into the stem cell. (D) During development, murine epidermal progenitors balance ACDs and SCDs to stratify the skin. Basal progenitors adhere to the basement membrane (niche) through β-integrins (green), and to neighboring cells through adherens junctions (magenta rods). These contacts and the apical localization of the Par complex Par3/Par6/aPKC (purple dots) define the progenitor apico-basal polarity. Vertical ACDs (left) occur with the spindle aligned to the apico-basal polarity axis, and generate a basal progenitor and a differentiating suprabasal cell inheriting Par3, Insc, LGN, and NuMA (cyan dots). Planar SCDs expand the basal progenitor pool (right). (E) During hair follicle (HF) morphogenesis (top panel), HFSCs originate by ACDs of epithelial placode cells. These cells divide perpendicular to the tissue basement membrane with LGN (cyan dots) partitioned into the suprabasal cell, and integrins (green) and Wnt components confined in the basal cell. In the adult hair follicle (bottom panel), mesenchymal cells lying beneath the placode condense in the dermal papilla (DP) with niche functions. HFSCs show a dual localization: quiescent HFSCs in the bulge and activated HFSCs in the hair germ in direct contact with the DP. Activated HFSCs divide perpendicularly to the niche, generating the inner differentiated layers (gray area), whereas undifferentiated HFSCs expand in the outer layer by oriented divisions. (F) The small intestine is formed by a monolayered epithelium folding into villi and crypts. At the crypt base, ISCs intercalate with Paneth cells (green) secreting Wnt ligands and thus acting as niche. Upon proliferation, ISCs move upward along the crypt wall, experience reduced Wnt signals, and differentiate into transit-amplifying (TA) progenitors. TA progenitors, in turn, differentiate into the variety of cells that populate the villi to replace the epithelial cells which are shed into the intestinal lumen at the villus tip. Download figure Download PowerPoint The connection between deregulated stem cell proliferation and tumor biology is one of the major discoveries of the last decade 1. Seminal studies in Drosophila larval brains revealed that aberrant symmetric divisions and defective cell cycle exit of mutated neuroblasts suffice to generate massive tumor-like overgrowth 23. In vertebrates, an equally clear demonstration that switching from asymmetric to symmetric cell divisions is sufficient to cause cancer is still lacking, likely due to technical difficulties in identifying correctly stem cells and studying their proliferation and differentiation potential, as well as to our limited knowledge of ACDs in vertebrates. Nonetheless, converging evidence indicates that in several human cancers, aggressiveness correlates with a stem cell signature, and expansion of stem cell compartments causes tissue disorganization and malignant overproliferation 4. In this review, we summarize the principles underlying the execution of ACDs highlighting the specific aspects of asymmetry addressed by the assays most commonly used to study stem cells (see Box 1 and Fig 2 for a summary of assays used). In addition, we survey the experimental approaches used to uncover the molecular contribution of cancer stem cells (CSCs) to tumor progression, with particular emphasis on the role of mammary stem cell ACDs in breast cancer. Box 1: Experimental assays employed to study asymmetric divisions Imaging According to a mechanistic definition, ACDs deal with the unequal partitioning of fate determinants and niche contacts. Thus, monitoring the orientation of division and the partitioning of such determinants can be considered the best indication of asymmetry, when—and only when—the identity of the niche and the determinants is clearly defined for the system under investigation. Because mitosis is a dynamical process, the best insights into the mechanisms of ACD have been obtained by live imaging, in which the distribution of determinants and the position of daughter cells after cytokinesis can be followed both in space and in time 1842146. Lineage tracing In lineage tracing experiments, cells expressing a SC-specific promoter are tagged with a permanent and heritable genetic marker. Most commonly, reporter genes for the expression of GFP, RFP, YFP, mCherry genes are used. The distribution of the marker is monitored long term within a tissue or an entire organism, ideally in vivo. If all the differentiated lineages can be tracked back to a single cell, this cell can be considered a multipotent stem cell. Long-term generation of marked cell lineages indicates that the labeled cell has self-renewal capacity. Sphere-forming assays The ability of stem cells isolated from tissues and grown in non-adherent cultures to form clonal spheroids when plated in liquid or semi-liquid cultures has been used to identify the number of stem cells within a cell population. The assay is based on the notion that only cells with SC properties are able to form a spheroid composed of cells at different differentiation stages, as assessed by functional assays. Therefore, the number of spheres in culture reflects the number of stem cells. In the case of MaSCs, these spheres are called mammospheres. Mammospheres can be serially passaged and the sphere-forming efficiency (SFE) expressed as a measure of their self-renewal potential. SCs dividing mainly asymmetrically will progressively decrease their SFE until complete exhaustion in culture, whereas SCs dividing mainly symmetrically will aberrantly expand and propagate indefinitely. SCs and progenitors within mammospheres can be isolated to near homogeneity using the PKH26-based label retention assay. PKH26 is a lipophilic dye that labels the cell membrane and gets segregated upon cellular divisions. Cells that are quiescent or slowly dividing are enriched in MaSCs (~1:4) and retain the label during the assay. PKH26 retention has been employed for determination of the mode of SC division, as ACDs generate one cell that remains quiescent, and therefore retains the label, and another that continues to divide. In a culture in which SCDs are prevalent, instead, the dye will be also rapidly diluted in MaSCs. Indeed, in this case, the PKHneg fraction of cells will be able to initiate a mammosphere culture and re-form a mammary gland upon transplantation 667072. Organoids Primary stem cells purified from a number of tissues including intestine, pancreas, liver, and brain can be expanded in clonal 3D organoids with architectural features resembling the organ of origin. The differentiation pattern sustaining organoid morphogenesis can be ascribed to self-renewing ACDs, which indeed have been proven experimentally in the case of cerebral organoids by lineage tracing experiments 147. Organ reconstitution upon transplantation The ability to reconstitute a tissue in a recipient host has been widely used to assess the presence of SCs in a given population. In the mammary system, cells are transplanted in the gland of pre-pubertal mice ablated of the rudimentary epithelial branches, and the presence of a fully differentiated mammary outgrowth is assessed 10–14 weeks after transplant. The transplantation protocol in limiting dilution conditions requires injecting a serially diluted number of cells into the cleared fat pad of pre-pubertal recipient mice. This allows to measure the size of the stem cell pool in a given population and to assess whether one single stem cell might be sufficient to reconstitute the gland tissue. In this setting, it is indirectly assumed that rounds of ACDs and, eventually, SCDs are needed to balance self-renewal and expansion. The calculated frequency of SCs reflects the prevalence of one of the two modes of division 70. Serial transplantation, instead, functionally defines stem cells for their ability to self-renew by generating new stem cells alongside a more differentiated progeny 148. In theory, asymmetric stem cell self-renewal ensures maintenance of a stable number of stem cells. However, wild-type stem cells become functionally exhausted after six to seven divisions; therefore, a constant (in numbers) stem cell pool has limited organ reconstitution ability upon serial transplantation. If instead the stem cell pool expands through symmetric divisions, then an infinite number of in vivo passages can be achieved 70. Figure 2. Schematic representation of the assays used to study ACDs in mammary stem cells Details of the working principles and the kind of information provided by each of the assays are reported in Box 1. (A) Imaging: Visualization of the distribution of DNA and fate determinants with respect to a known cellular niche allows monitoring of asymmetric fate partitioning and daughter cell positioning. Live imaging of the mitotic process is most informative. (B) Lineage tracing: Transgenic mice expressing an inducible Cre recombinase (Cre-ER) under the control of a stem cell-specific promoter are crossed with a reporter model harboring a stop codon flanked by LoxP sites upstream of a reporter gene (e.g., GFP in the figure) under a constitutive promoter. Administration of 4-hydroxytamoxifen (4-OHT) allows the activation of the Cre in cells expressing the SC promoter. Cre mediates the recombination between the LoxP sites, causing the excision of the stop cassette and leading to the permanent expression of the reporter gene in the SC and its progeny. In order to minimize any adverse effects of the 4-OHT administration on the mammary gland, the Cre gene can also be expressed under a Tet-ON/OFF system of inducible regulation. (C) Top: Sphere-forming assay. Epithelial cells isolated from the mammary gland can be grown in anchorage-independent conditions allowing the formation of mammospheres. Mammospheres are clonal in origin, contain SCs and more differentiated cells (progenitors), and can be serially passaged. Sphere-forming efficiency (SFE) is calculated as a percentage of total number of cells plated (the number of spheres formed/the number of cells plated). Bottom: PKH26 assay. Epithelial cells are labeled with the lipophilic dye PKH26 and allowed to grow as mammospheres for label retention. Only the quiescent or slowly dividing cells will retain PKH26 at the end of the assay. In a culture in which SCDs are prevalent, the PKHneg population will contain cells with SC features, able to form mammospheres and positive transplantations. (D) Organoids: Isolated and digested mammary epithelium is embedded in Matrigel supplemented with ECM components that allow branching. (E) Transplantation assay: Isolated epithelial cells are transplanted in the cleared fat pad of pre-pubertal recipients and their ability to reconstitute a mammary gland is assessed. Transplantation in limiting dilution conditions allows calculating SC frequency, while serial transplantation allows measuring SC lifespan. Both assays provide information regarding the size of the SC pool in a given population. Download figure Download PowerPoint Operational definition of ACDs: basic lessons from flies The concept of self-renewal as defined in the previous section implies that stem cells enter the cell cycle and divide, and that at least one of the progeny is an undifferentiated cell identical to the mother. Notably, short-term self-renewal has also been documented in progenitors, thus complicating the design of experimental assays aimed at studying stem cell ACDs based on their proliferation potential. Furthermore, stem cell divisions are accompanied by both periods of quiescence and maturation events occurring after completion of the self-renewing mitosis. These non-mitotic processes, which in a physiological context are strictly connected with self-renewing mitoses of stem cells, will not be discussed in this review. Unequal partitioning of polarity proteins and cell fate determinants Much has been learnt in recent years on the molecular mechanisms of ACDs. Studies conducted in the early 1990 in Drosophila neuroblasts (NBs, the stem cells of the central nervous system), sensory organ precursors (or SOPs, precursors of the peripheral nervous systems), and male germline stem cells paved the way to the identification of fundamental principles of ACDs. Neuroblasts always divide asymmetrically to give rise to one neuroblast and one ganglion mother cell, the latter destined to differentiate into two neurons or glia (Fig 1B). The remarkable ability of neuroblasts to undergo ACDs even in isolated cultures has made them the prototype of intrinsic ACDs, which are characterized by the ability of mitotic cells to cell-autonomously polarize the cortex, align the mitotic spindle along the polarity axis, and unequally partition cellular components, including the polarity proteins Par3/Par6/aPKC and the so-called fate determinants, that is, molecules able to confer a given fate on the cell that inherits them. In neuroblasts, this activity has been documented for the transcription factor Prospero, the endocytic Notch inhibitor Numb, and the tumor suppressor Brat 56. The characterization of genetic lesions impairing asymmetry in neuroblasts also led to the identification of genes coding for proteins essential for mitotic spindle coupling with cortical polarity, such as the Par3-binding protein Inscuteable 7, the switch molecule Pins/Rapsinoid, the Dynein-adaptor Mud 389, the Aurora-A kinase 101112, and the Gαi subunit of heterotrimeric G-proteins 1314 (see Table 1 for a description of their functions). Most notably, the asymmetric distributions of Par3, aPKC, Pins (LGN in vertebrates), and Numb with respect to the orientation of the division plane are often regarded as the distinguishing feature of ACDs. However, it is important to stress that it has never been formally proven that these molecules act ubiquitously as fate determinants. Table 1. Names, functions, and interactions of proteins involved in ACD as discussed in this review Drosophila Vertebrates Protein type/epithelial localization/function Bazooka Par3/Par3L Polarity protein/apical (TJs)/polarity establishment Par6 Par6 Polarity protein/apical/polarity establishment aPKC aPKCζ/ι/λ Polarity protein, Ser/Thr kinase/apical/polarity establishment Lkb1 Lkb1 Ser/Thr kinase/apical/proliferation Scrib Scrib Scaffold protein/baso-lateral/polarity regulator Dlg1 DLG1 Scaffold protein/baso-lateral/polarity regulator Lgl Llgl Scaffold protein/baso-lateral/polarity regulator Inscuteable mInsc SC adaptor molecule/apical/orientation; partner of Par3 and LGN Pins LGN Scaffolding protein/apical (ACD), lateral (SCD)/orientation; partner of mInsc and NuMA Mud NuMA Adaptor/apical (ACD), lateral (SCD) and at spindle poles/orientation; partner of LGN and Dynein Gαi/Gα0 Gαi Subunit of heterotrimeric G-proteins/plasma membrane/orientation; partner of LGN Dynein Dynein MT motor/apical (ACD), lateral (SCD)/orientation; partner of NuMA − β1-integrin Collagen receptor/basal membrane/niche contact, orientation Huntingtin Huntingtin MT-associating protein/spindle poles/orientation, partner of Dynein Prospero Prox1 Transcription factor/fate determinant − Lef1 Transcription factor/Wnt signaling effector, differentiation Armadillo β-catenin Transcriptional co-activator and adhesion/Wnt signaling effector; partner of Lef1 − p53 Transcription factor/apoptosis, senescence, DNA damage response − SOX-9 Transcription factor/SHH signaling effector, stemness Numb Numb Endocytic Notch inhibitor/basal (Dm neuroblasts)/fate determinant Brat Trim32 Scaffolding protein/basal (Dm neuroblasts)/fate determinant − Musashi RNA-binding protein/regulation of Numb translation − miR-34a microRNA/Numb mRNA targeting − ErbB2/Her2 Tyrosine kinase receptor/lateral membrane/proliferation Aurora-A Aurora-A Ser/Thr kinase/spindle poles/orientation, bipolar spindle assembly Proteins are listed based on their role; from the top: polarity proteins, spindle orientation components, and fate determinants. In parallel, recent research consolidated the view that stem cell maintenance depends on specific master transcriptional regulators, whose activity is governed by asymmetrically segregating epigenetic marks. Using a photoconvertible dual-color method, it was possible to demonstrate that during male germline stem cell (GSC hereon) asymmetric divisions, the pre-existing pool of histone H3 selectively segregates into the GSCs thanks to selective phosphorylation on H3-Thr10 by the mitotic kinase Haspin 15, whereas the newly synthesized H3, incorporated into nucleosomes during DNA replication, partitions into the differentiating cell 16. Unequal sister cell positioning with respect to the niche In flies, also GSC ACDs occur with a stereotypical orientation with respect to cortical polarity. In this case, however, asymmetry is not cell autonomous as for NBs, but relies on the GSC microenvironment (Fig 1C). GSCs reside in a specialized niche, physically attached to somatic hub cells in an E-cadherin-dependent manner 17. Hub cells secrete the ligand Unpaired that activates Jak-STAT signaling in the neighboring cells, in this way maintaining their stem cell identity. GSCs divide asymmetrically with the spindle perpendicular to the hub so that only one daughter retains contact with the hub cells, and hence stemness. Interestingly, upon stem cell loss, vacancies are replenished by proliferative symmetric divisions which are oriented parallel to the hub. Elegant live imaging studies revealed that GSC perpendicular divisions occasionally result in the production of two GSCs or two differentiating cells, and that upon injuries differentiated cells can revert to GSCs by migrating back to the hub 18, thus displaying a cell plasticity impossible to observe in fixed tissues. The fly GSC behavior well exemplifies the concept of stem cell niche, a “specific anatomic location that regulates how stem cells participate in tissue regeneration, maintenance, and repair” 19. It also highlights the paramount importance of sister cell positioning with respect to the niche for fate specification. Alignment of the mitotic spindle with the polarity axis Studies in neuroblasts and GSCs uncovered an intimate connection between the orientation of the division plane (and hence the spindle axis) and the asymmetry of fate specification, which is required for the asymmetric segregation of both determinants and adhesion molecules/cell–cell contacts. Elegant studies with molecular markers specific for mother and daughter centrioles revealed that in these systems, not only ACDs occur with the mitotic spindle aligned with the polarity axis and perpendicular to the niche, but also that mother and daughter centrosomes are specifically partitioned either in the stem or in the differentiating cell 2021, suggesting that the inherent centrosome asymmetrical maturation is involved in asymmetric fate choices. Also, the asymmetric outcome of SOP divisions relies on tissue-derived external cues that instruct the anterior–posterior cortical polarity, though in a different flavor of environmental mitotic regulation compared to niches as defined before 14. SOP cells delaminate from a polarized epithelium, from which they inherit planar polarity that is used in two subsequent mitoses to asymmetrically segregate Notch regulators (including Numb) and to generate two outer and two inner sensory cells. Because of this simple lineage, the precise knowledge of fate determinants, and the easiness of imaging, SOPs have provided numerous insights into molecular mechanisms of asymmetry. Segalen et al 22 showed that the non-canonical Wnt pathway is involved in establishing correct SOP orientation by direct binding of the spindle motors to the Wnt-effector disheveled. More recently, it was also shown that specialized endosomes, known as Sara endosomes, are inherited asymmetrically by the two SOP daughter cells, PIIa and PIIb, and mediate Notch/Delta signaling between the nascent sisters 23 via the action of antagonistic kinesins moving on the central spindle during cytokinesis 24. Importantly, these latter studies revealed that organelle trafficking is implicated in ACDs. In addition to these mechanistic insights into how ACDs are executed, fly neuroblasts served as a model tool to explore the effect of the aberrant switch between asymmetric and symmetric divisions on the stem cell proliferative potential. Seminal studies from the Gonzalez laboratory not only revealed that impairment of asymmetry-related genes causes massive expansion of the neuroblast compartment, but also that, upon allograft implantation, asymmetry-defective neuroblasts hyper-proliferate into wild-type fly abdomens and subvert tissue homeostasis in a malignant-like form 2. Thus, these assays first highlighted the intimate causative connection between stem cell deregulation and cancer. ACDs in vertebrates Studies of ACDs in vertebrate systems lagged substantially behind the ones in flies, mainly because of imaging difficulties and lack of stem cell markers amenable to track asymmetric divisions. Contrary to fly neuroblasts, vertebrate stem cells balance SCDs versus ACDs in response to developmental programs and regenerative stimuli. Recent studies have revealed that in vertebrates, the symmetric or asymmetric outcome of each individual SC division is unpredictable. Nonetheless, the ratio between the two modes of division within a stem cell population is tightly regulated in order to maintain tissue homeostasis, and it plastically readjusts upon injuries in order to promote regeneration. Notably, this intrinsic stochasticity in differentiation may also account for clonal competition of mutant stem cells and therefore might contribute to cancer development 25. Molecularly, extracellular cues instructing stem cell divisions are communicated as short-range morphogen gradients such as Wnt, Hedgehog (HH), EGF and BMP signaling 26. For these reasons, studies of mitotic phenotypes of vertebrate stem cells require in vivo population statistics and greatly benefited from recent developments of intravital microscopy 27. Often, experimental strategies addressing ACDs in vertebrates have been guided by working principles learnt in Drosophila stem cells, somehow biasing the discovery of novel and more specific mechanisms. In this paragraph, we will review the approaches used to study ACDs in vertebrates, emphasizing whether asymmetry was approached from a mechanistic perspective, investigating the self-renewing mitosis, or by lineage tracing of daughter cells. In particular, we will discuss stem cells of the epidermis and intestinal crypts, which thanks to their fast cycling rates and accessibility have been easier to characterize. Breast stem cells will be discussed in more detail in following sections. Epidermal stem cells: contact with the basement

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