Molecular mechanisms of asymmetric divisions in mammary stem cells

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.

Keywords: asymmetric cell division assays; asymmetric cell divisions; breast cancer; mammary stem cells.

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.

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 PKH ^neg 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.

Figure 3. Asymmetric self‐renewal in mammary gland morphogenesis

(A) Schematic view of a post‐pubertal fat pad in which the mammary epithelium is embedded. The ductal epithelium is composed by a basal/myoepithelial layer, which is in contact with the basement membrane, and a luminal layer with secreting function. Lineage tracing experiments have demonstrated the existence of bipotent MaSCs residing in the basal layer 69 and of unipotent basal and luminal SCs 67 (white cells). The ducts are surrounded by fibroblasts, collagen fibrils, and circulating cells of the immune system, all embedded in the adipose tissue. The invasion of the fat pad starts at puberty from the TEBs. The TEB is considered a SC niche where cap cells and body cells highly proliferate constituting the leading edge of the invasion front. The cap cell layer contains MaSCs (white cells) that are able to undergo symmetric and asymmetric self‐renewal 72, 75, 95. Cap cells' mitotic divisions taking place perpendicularly to the basement membrane are asymmetric, as one of the daughter cell abandons the contacts with the original niche (dividing cells at the bottom of the bud). Cap cells' mitotic divisions parallel to the basement membrane are considered symmetric and guarantee maintenance of SC identity and expansion of SC number (dividing cells at the top of the bud) 95, 99, 100. It is not known whether parallel division could also result in asymmetric fate and lead to the generation of more differentiated cap cells (dividing cells on the right side of the bud). (B) Stages of mammary gland development. At birth, the mammary epithelium is only rudimentary and remains quiescent until puberty. During puberty, hormonal cues stimulate the formation of TEBs and the elongation, bifurcation, and side‐branching of the mammary tree. The gland reaches its full development and functional specialization upon pregnancy when alveologenesis and lactogenesis take place. After lactation, the epithelium involutes and returns to a state that resembles that of the virgin gland. (C) Mammary epithelium cell hierarchy. Schematic summary of the available markers used to isolate distinct subsets of mammary cells at different differentiation stages.