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Notch1-Dll4 signalling and mechanical force regulate leader cell formation during collective cell migration.

Riahi R, Sun J, Wang S, Long M, Zhang DD, Wong PK - Nat Commun (2015)

Bottom Line: However, the factors driving the leader cell formation as well as the mechanisms regulating leader cell density during the migration process remain to be determined.Furthermore, mechanical stress inhibits Dll4 expression and leader cell formation in the monolayer.Collectively, our findings suggest that a reduction of mechanical force near the boundary promotes Notch1-Dll4 signalling to dynamically regulate the density of leader cells during collective cell migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Aerospace and Mechanical Engineering, The University of Arizona, Tucson, Arizona 85721-0119, USA.

ABSTRACT
At the onset of collective cell migration, a subset of cells within an initially homogenous population acquires a distinct 'leader' phenotype with characteristic morphology and motility. However, the factors driving the leader cell formation as well as the mechanisms regulating leader cell density during the migration process remain to be determined. Here we use single-cell gene expression analysis and computational modelling to show that the leader cell identity is dynamically regulated by Dll4 signalling through both Notch1 and cellular stress in a migrating epithelium. Time-lapse microscopy reveals that Dll4 is induced in leader cells after the creation of the cell-free region and leader cells are regulated via Notch1-Dll4 lateral inhibition. Furthermore, mechanical stress inhibits Dll4 expression and leader cell formation in the monolayer. Collectively, our findings suggest that a reduction of mechanical force near the boundary promotes Notch1-Dll4 signalling to dynamically regulate the density of leader cells during collective cell migration.

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Characteristics of leader cells in collective cell migration(a) Schematic representation of a migration tip with leader-follower organization during collective cell migration. Leader cells (green) at the front of the leading edge typically display enlarged cell size, ruffling lamellipodium, large focal adhesions and aligned cytoskeletal architecture. (b) Representative immunofluorescence image of F-actin (red) and vinculin (green) in leader cells formed at the leading edge. (c-e) Actin stress fibers in cells transfected with double-stranded locked nucleic acid (dsLNA) probes targeting (c) β-actin mRNA, (d) Dll4 mRNA, and (e) a random sequence. Cells were first transfected with the dsLNA probes (green) and fixed for immunostaining (red). (f-h) Focal adhesion in cells transfected with dsLNA probes targeting β-actin mRNA (f), Dll4 mRNA (g), and a random sequence (h). Samples were counterstained with DAPI (blue). Images are representative of three independent experiments. Scale bars, 50 μm. (i-j) HE staining of epithelial cells in skin punch wounds. (k-m) IHC staining of IgG control (k), Dll4 (l), and Notch1 (m). Yellow dotted lines indicate the epithelium and black arrows indicate the wound boundaries. Scale bars, 200 μm.
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Figure 1: Characteristics of leader cells in collective cell migration(a) Schematic representation of a migration tip with leader-follower organization during collective cell migration. Leader cells (green) at the front of the leading edge typically display enlarged cell size, ruffling lamellipodium, large focal adhesions and aligned cytoskeletal architecture. (b) Representative immunofluorescence image of F-actin (red) and vinculin (green) in leader cells formed at the leading edge. (c-e) Actin stress fibers in cells transfected with double-stranded locked nucleic acid (dsLNA) probes targeting (c) β-actin mRNA, (d) Dll4 mRNA, and (e) a random sequence. Cells were first transfected with the dsLNA probes (green) and fixed for immunostaining (red). (f-h) Focal adhesion in cells transfected with dsLNA probes targeting β-actin mRNA (f), Dll4 mRNA (g), and a random sequence (h). Samples were counterstained with DAPI (blue). Images are representative of three independent experiments. Scale bars, 50 μm. (i-j) HE staining of epithelial cells in skin punch wounds. (k-m) IHC staining of IgG control (k), Dll4 (l), and Notch1 (m). Yellow dotted lines indicate the epithelium and black arrows indicate the wound boundaries. Scale bars, 200 μm.

Mentions: A known mechanism of collective migration is “purse string” closure, where a multicellular actin bundle formed among cells at the boundary of a small wound draws the wound together8, 9. Another mechanism of collective cell migration is the formation of migration tips3 (Fig. 1a). Migration tips with leader-follower organization are observed in the healing of larger wounds and cancer invasion. In particular, specialized leader cells appear at the leading edge and exert mechanical force on follower cells10, 11. In an organotypic co-culture invasion model, fibroblasts serve as leaders to drive the collective migration of carcinoma cells12. Remarkably, integrins α3 and α5 along with myosin light chain activity in fibroblasts are required for force-mediated matrix remodeling; however, these factors are not required in the trailing carcinomas, suggesting biomechanical coupling and a leader-follower organization in the invasion process.


Notch1-Dll4 signalling and mechanical force regulate leader cell formation during collective cell migration.

Riahi R, Sun J, Wang S, Long M, Zhang DD, Wong PK - Nat Commun (2015)

Characteristics of leader cells in collective cell migration(a) Schematic representation of a migration tip with leader-follower organization during collective cell migration. Leader cells (green) at the front of the leading edge typically display enlarged cell size, ruffling lamellipodium, large focal adhesions and aligned cytoskeletal architecture. (b) Representative immunofluorescence image of F-actin (red) and vinculin (green) in leader cells formed at the leading edge. (c-e) Actin stress fibers in cells transfected with double-stranded locked nucleic acid (dsLNA) probes targeting (c) β-actin mRNA, (d) Dll4 mRNA, and (e) a random sequence. Cells were first transfected with the dsLNA probes (green) and fixed for immunostaining (red). (f-h) Focal adhesion in cells transfected with dsLNA probes targeting β-actin mRNA (f), Dll4 mRNA (g), and a random sequence (h). Samples were counterstained with DAPI (blue). Images are representative of three independent experiments. Scale bars, 50 μm. (i-j) HE staining of epithelial cells in skin punch wounds. (k-m) IHC staining of IgG control (k), Dll4 (l), and Notch1 (m). Yellow dotted lines indicate the epithelium and black arrows indicate the wound boundaries. Scale bars, 200 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4380165&req=5

Figure 1: Characteristics of leader cells in collective cell migration(a) Schematic representation of a migration tip with leader-follower organization during collective cell migration. Leader cells (green) at the front of the leading edge typically display enlarged cell size, ruffling lamellipodium, large focal adhesions and aligned cytoskeletal architecture. (b) Representative immunofluorescence image of F-actin (red) and vinculin (green) in leader cells formed at the leading edge. (c-e) Actin stress fibers in cells transfected with double-stranded locked nucleic acid (dsLNA) probes targeting (c) β-actin mRNA, (d) Dll4 mRNA, and (e) a random sequence. Cells were first transfected with the dsLNA probes (green) and fixed for immunostaining (red). (f-h) Focal adhesion in cells transfected with dsLNA probes targeting β-actin mRNA (f), Dll4 mRNA (g), and a random sequence (h). Samples were counterstained with DAPI (blue). Images are representative of three independent experiments. Scale bars, 50 μm. (i-j) HE staining of epithelial cells in skin punch wounds. (k-m) IHC staining of IgG control (k), Dll4 (l), and Notch1 (m). Yellow dotted lines indicate the epithelium and black arrows indicate the wound boundaries. Scale bars, 200 μm.
Mentions: A known mechanism of collective migration is “purse string” closure, where a multicellular actin bundle formed among cells at the boundary of a small wound draws the wound together8, 9. Another mechanism of collective cell migration is the formation of migration tips3 (Fig. 1a). Migration tips with leader-follower organization are observed in the healing of larger wounds and cancer invasion. In particular, specialized leader cells appear at the leading edge and exert mechanical force on follower cells10, 11. In an organotypic co-culture invasion model, fibroblasts serve as leaders to drive the collective migration of carcinoma cells12. Remarkably, integrins α3 and α5 along with myosin light chain activity in fibroblasts are required for force-mediated matrix remodeling; however, these factors are not required in the trailing carcinomas, suggesting biomechanical coupling and a leader-follower organization in the invasion process.

Bottom Line: However, the factors driving the leader cell formation as well as the mechanisms regulating leader cell density during the migration process remain to be determined.Furthermore, mechanical stress inhibits Dll4 expression and leader cell formation in the monolayer.Collectively, our findings suggest that a reduction of mechanical force near the boundary promotes Notch1-Dll4 signalling to dynamically regulate the density of leader cells during collective cell migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Aerospace and Mechanical Engineering, The University of Arizona, Tucson, Arizona 85721-0119, USA.

ABSTRACT
At the onset of collective cell migration, a subset of cells within an initially homogenous population acquires a distinct 'leader' phenotype with characteristic morphology and motility. However, the factors driving the leader cell formation as well as the mechanisms regulating leader cell density during the migration process remain to be determined. Here we use single-cell gene expression analysis and computational modelling to show that the leader cell identity is dynamically regulated by Dll4 signalling through both Notch1 and cellular stress in a migrating epithelium. Time-lapse microscopy reveals that Dll4 is induced in leader cells after the creation of the cell-free region and leader cells are regulated via Notch1-Dll4 lateral inhibition. Furthermore, mechanical stress inhibits Dll4 expression and leader cell formation in the monolayer. Collectively, our findings suggest that a reduction of mechanical force near the boundary promotes Notch1-Dll4 signalling to dynamically regulate the density of leader cells during collective cell migration.

Show MeSH
Related in: MedlinePlus