Alexandra Van Keymeulen

Distinct stem cells contribute to mammary gland development and maintenance

The mammary epithelium is composed of several cell lineages including luminal, alveolar and myoepithelial cells. Transplantation studies have suggested that the mammary epithelium is maintained by the presence of multipotent mammary stem cells. To define the cellular hierarchy of the mammary gland during physiological conditions, we performed genetic lineage-tracing experiments and clonal analysis of the mouse mammary gland during development, adulthood and pregnancy. We found that in postnatal unperturbed mammary gland, both luminal and myoepithelial lineages contain long-lived unipotent stem cells that display extensive renewing capacities, as demonstrated by their ability to clonally expand during morphogenesis and adult life as well as undergo massive expansion during several cycles of pregnancy. The demonstration that the mammary gland contains different types of long-lived stem cells has profound implications for our understanding of mammary gland physiology and will be instrumental in unravelling the cells at the origin of breast cancers.

Identification of the cell lineage at the origin of basal cell carcinoma.

For most types of cancers, the cell at the origin of tumour initiation is still unknown. Here, we used mouse genetics to identify cells at the origin of basal cell carcinoma (BCC), which is one of the most frequently occurring types of cancer in humans, and can result from the activation of the Hedgehog signalling pathway. Using mice conditionally expressing constitutively active Smoothened mutant (SmoM2), we activated Hedgehog signalling in different cellular compartments of the skin epidermis and determined in which compartments Hedgehog activation induces BCC formation. Activation of SmoM2 in hair follicle bulge stem cells and their transient amplifying progenies did not induce cancer formation, demonstrating that BCC does not originate from bulge stem cells, as previously thought. Using clonal analysis, we found that BCC arises from long-term resident progenitor cells of the interfollicular epidermis and the upper infundibulum. Our studies uncover the cells at the origin of BCC in mice and demonstrate that expression of differentiation markers in tumour cells is not necessarily predictive of the cancer initiating cells.

To stabilize neutrophil polarity, PIP3 and Cdc42 augment RhoA activity at the back as well as signals at the front

Chemoattractants like f-Met-Leu-Phe (fMLP) induce neutrophils to polarize by triggering divergent signals that promote the formation of protrusive filamentous actin (F-actin; frontness) and RhoA-dependent actomyosin contraction (backness). Frontness locally inhibits backness and vice versa. In neutrophil-like HL60 cells, blocking phosphatidylinositol-3,4,5-tris-phosphate (PIP3) accumulation with selective inhibitors of PIP3 synthesis completely prevents fMLP from activating a PIP3-dependent kinase and Cdc42 but not from stimulating F-actin accumulation. PIP3-deficient cells show reduced fMLP-dependent Rac activity and unstable pseudopods, which is consistent with the established role of PIP3 as a mediator of positive feedback pathways that augment Rac activation at the front. Surprisingly, such cells also show reduced RhoA activation and RhoA-dependent contraction at the trailing edge, leading to the formation of multiple lateral pseudopods. Cdc42 mediates PIP3s positive effect on RhoA activity. Thus, PIP3 and Cdc42 maintain stable polarity with a single front and a single back not only by strengthening pseudopods but also, at longer range, by promoting RhoA-dependent actomyosin contraction at the trailing edge.

Multipotent and unipotent progenitors contribute to prostate postnatal development.

The prostate is a glandular epithelium composed of basal, luminal and neuroendocrine cells that originate from the urogenital sinus during embryonic development. After birth, the prostate keeps developing until the end of puberty. Here, we used inducible genetic lineage tracing experiments in mice to investigate the cellular hierarchy that governs prostate postnatal development. We found that prostate postnatal development is mediated by basal multipotent stem cells that differentiate into basal, luminal and neuroendocrine cells, as well as by unipotent basal and luminal progenitors. Clonal analysis of basal cells revealed the existence of bipotent and unipotent basal progenitors as well as basal cells already committed to the luminal lineage with intermediate cells co-expressing basal and luminal markers associated with this commitment step. The existence of multipotent basal progenitors during prostate postnatal development contrasts with the distinct pools of unipotent basal and luminal stem cells that mediate adult prostate regeneration. Our results uncover the cellular hierarchy acting during prostate development and will be instrumental in defining the cellular origin and the mechanisms underlying prostate cancer initiation.

Polarity reveals intrinsic cell chirality

Like blood neutrophils, dHL60 cells respond to a uniform concentration of attractant by polarizing in apparently random directions. How each cell chooses its own direction is unknown. We now find that an arrow drawn from the center of the nucleus of an unpolarized cell to its centrosome strongly predicts the subsequent direction of attractant-induced polarity: Of 60 cells that polarized in response to uniform f-Met-Leu-Phe (fMLP), 42 polarized to the left of this arrow, 6 polarized to the right, and 12 polarized directly toward or away from the centrosome. To investigate this directional bias we perturbed a regulatory pathway, downstream of Cdc42 and partitioning-defective 6 (Par6), which controls centrosome orientation relative to polarity of other cells. Dominant negative Par6 mutants block polarity altogether, as previously shown for disrupting Cdc42 activity. Cells remain able to polarize, but without directional bias, if their microtubules are disrupted with nocodazole, or they express mutant proteins that interfere with activities of PKCζ or dynein. Expressing constitutively active glycogen synthase kinase 3β (GSK3β) causes cells to polarize preferentially to the right. Distributions of most of these polarity regulators localize to the centrosome but show no left–right asymmetry before polarization. Together, these findings suggest that an intrinsically chiral structure, perhaps the centrosome, serves as a template for directing polarity in the absence of spatial cues. Such a template could help to determine left–right asymmetry and planar polarity in development.

The long noncoding RNA Neat1 is required for mammary gland development and lactation

The lncRNA Neat1 is an essential architectural component of paraspeckle nuclear bodies. Although cell-based studies identified Neat1-paraspeckles as key regulators of gene expression through retention of hyperdited mRNAs and/or transcription factors, it is unclear under which specific physiological conditions paraspeckles are formed in vivo and whether they have any biological relevance. Herein, we show that paraspeckles are assembled in luminal epithelial cells during mammary gland development. Importantly, genetic ablation of Neat1 results in aberrant mammary gland morphogenesis and lactation defects. We provide evidence that the lactation defect is caused by a decreased ability of Neat1-mutant cells to sustain high rates of proliferation during lobular-alveolar development. This study is the first to assign an important biological function to the lncRNA Neat1 and to link it to the presence of paraspeckles nuclear bodies in vivo.

PDZRhoGEF and myosin II localize RhoA activity to the back of polarizing neutrophil-like cells.

Chemoattractants such as formyl-Met-Leu-Phe (fMLP) induce neutrophils to polarize by triggering divergent pathways that promote formation of a protrusive front and contracting back and sides. RhoA, a Rho GTPase, stimulates assembly of actomyosin contractile complexes at the sides and back. We show here, in differentiated HL60 cells, that PDZRhoGEF (PRG), a guanine nucleotide exchange factor (GEF) for RhoA, mediates RhoA-dependent responses and determines their spatial distribution. As with RNAi knock-down of PRG, a GEF-deleted PRG mutant blocks fMLP-dependent RhoA activation and causes neutrophils to exhibit multiple fronts and long tails. Similarly, inhibition of RhoA, a Rho-dependent protein kinase (ROCK), or myosin II produces the same morphologies. PRG inhibition reduces or mislocalizes monophosphorylated myosin light chains in fMLP-stimulated cells, and myosin II ATPase inhibition reciprocally disrupts normal localization of PRG. We propose a cooperative reinforcing mechanism at the back of cells, in which PRG, RhoA, ROCK, myosin II, and actomyosin spatially cooperate to consolidate attractant-induced contractility and ensure robust cell polarity.

Tracing epithelial stem cells during development, homeostasis, and repair

Epithelia ensure many critical functions of the body, including protection against the external environment, nutrition, respiration, and reproduction. Stem cells (SCs) located in the various epithelia ensure the homeostasis and repair of these tissues throughout the lifetime of the animal. Genetic lineage tracing in mice has allowed the labeling of SCs and their progeny. This technique has been instrumental in characterizing the origin and heterogeneity of epithelial SCs, their tissue location, and their differentiation potential under physiological conditions and during tissue regeneration.

Clonal dynamics reveal two distinct populations of basal cells in slow turnover airway epithelium

Summary Epithelial lineages have been studied at cellular resolution in multiple organs that turn over rapidly. However, many epithelia, including those of the lung, liver, pancreas, and prostate, turn over slowly and may be regulated differently. We investigated the mouse tracheal epithelial lineage at homeostasis by using long-term clonal analysis and mathematical modeling. This pseudostratified epithelium contains basal cells and secretory and multiciliated luminal cells. Our analysis revealed that basal cells are heterogeneous, comprising approximately equal numbers of multipotent stem cells and committed precursors, which persist in the basal layer for 11 days before differentiating to luminal fate. We confirmed the molecular and functional differences within the basal population by using single-cell qRT-PCR and further lineage labeling. Additionally, we show that self-renewal of short-lived secretory cells is a feature of homeostasis. We have thus revealed early luminal commitment of cells that are morphologically indistinguishable from stem cells.

Cyclin D3 accumulation and activity integrate and rank the comitogenic pathways of thyrotropin and insulin in thyrocytes in primary culture.

The proliferation of most normal cells depends on the synergistic interaction of several growth factors and hormones, but the cell cycle basis for this combined requirement remains largely uncharacterized. We have addressed the question of the requirement for insulin/IGF-1 also observed in many cell culture systems in the physiologically relevant system of primary cultures of dog thyroid epithelial cells stimulated by TSH, which exerts its mitogenic activity only via cAMP. The induction of cyclin A and cdc2, the phosphorylation of cdk2, the nuclear translocation of cdk4 and the assembly of cyclin D3-cdk4 complexes required the synergy of TSH and insulin. Cyclin D3 (the most abundant cyclin D) was necessary for the proliferation stimulated by TSH in the presence of insulin as shown by microinjection of a neutralizing antibody. Cyclin D3 accumulation and activity were differentially regulated by insulin and TSH, which points out this cyclin as an integrator that ranks these comitogenic pathways as supportive and activatory, respectively. Paradoxically TSH alone strongly repressed cyclin D3 accumulation. This inhibition was overridden by insulin, which markedly stimulated cyclin D3 mRNA and protein accumulation, but failed to assemble cyclin D3-cdk4 complexes in the absence of TSH. TSH unmasked the DCS-22 epitope of cyclin D3 and assembled cyclin D3-cdk4 in the presence of insulin. These data demonstrate that cyclin D synthesis and cyclin D-cdk assembly can be dissociated and complementarily regulated by different agents and signalling pathways.