Collective Epithelial Migration Research Articles

Matrix micro-defects impede collective epithelial migration with collagen type-IV but not type-I

Matrix micro-defects impede collective epithelial migration with collagen type-IV but not type-I

Matrix obstructions cause multiscale disruption in collective epithelial migration by suppressing leader cell function.

During disease and development, physical changes in extracellular matrix cause jamming, unjamming, and scattering in epithelial migration. However, whether disruptions in matrix topology alter collective cell migration speed and cell-cell coordination remains unclear. We microfabricated substrates with stumps of defined geometry, density, and orientation, which create obstructions for migrating epithelial cells. Here, we show that cells lose their speed and directionality when moving through densely spaced obstructions. Although leader cells are stiffer than follower cells on flat substrates, dense obstructions cause overall cell softening. Through a lattice-based model, we identify cellular protrusions, cell-cell adhesions, and leader-follower communication as key mechanisms for obstruction-sensitive collective cell migration. Our modeling predictions and experimental validations show that cells' obstruction sensitivity requires an optimal balance of cell-cell adhesions and protrusions. Both MDCK (more cohesive) and α-catenin-depleted MCF10A cells were less obstruction sensitive than wild-type MCF10A cells. Together, microscale softening, mesoscale disorder, and macroscale multicellular communication enable epithelial cell populations to sense topological obstructions encountered in challenging environments. Thus, obstruction-sensitivity could define "mechanotype" of cells that collectively migrate yet maintain intercellular communication.

A unique form of collective epithelial migration is crucial for tissue fusion in the secondary palate and can overcome loss of epithelial apoptosis.

ABSTRACTTissue fusion frequently requires the removal of an epithelium that intervenes distinct primordia to form one continuous structure. In the mammalian secondary palate, a midline epithelial seam (MES) forms between two palatal shelves and must be removed to allow mesenchymal confluence. Abundant apoptosis and cell extrusion support their importance in MES removal. However, genetically disrupting the intrinsic apoptotic regulators BAX and BAK within the MES results in complete loss of cell death and cell extrusion, but successful removal of the MES. Novel static- and live-imaging approaches reveal that the MES is removed through streaming migration of epithelial trails and islands to reach the oral and nasal epithelial surfaces. Epithelial trail cells that express the basal epithelial marker ΔNp63 begin to express periderm markers, suggesting that migration is concomitant with differentiation. Live imaging reveals anisotropic actomyosin contractility within epithelial trails, and genetic ablation of actomyosin contractility results in dispersion of epithelial collectives and failure of normal MES migration. These findings demonstrate redundancy between cellular mechanisms of morphogenesis, and reveal a crucial and unique form of collective epithelial migration during tissue fusion.

Glycomics reveal that ST6GAL1‐mediated sialylation regulates uterine lumen closure during implantation

ObjectivesImplantation failure is a major cause of prenatal mortality. The uterine lumen closure contributes to embryo adhesion to the uterus, but its underlying mechanisms are largely unknown. Our previous study has reported that endometrial fold extension can lead to uterine lumen closure in pigs. The objective of this study was to reveal molecular mechanisms of the uterine lumen closure by characterizing the molecular basis of the endometrial fold extension during implantation in pigs.Materials and methodsUterine and endometrium tissues during implantation were collected in pigs. MALDI‐TOF MS was used to characterize the N‐glycomic profiles. Histochemistry, siRNA transfection, Western blotting, lectin immumoprecipitation, mass spectrometry and assays of wounding healing and cell aggregation were performed to investigate the molecular basis.ResultsWe observed that uterine luminal epithelium (LE) migrated collectively during endometrial fold extension. For the first time, we identified a large number of N‐glycan compositions from endometrium during implantation using MALDI‐TOF MS. Notably, the α2,6‐linked sialic acid and ST6GAL1 were highly expressed in uterine LE when the endometrial folds extended greatly. Subsequently, the role of ST6GAL1‐mediated 2,6‐sialylation in collective epithelial migration was demonstrated. Finally, we found that ST6GAL1‐mediated α2,6‐sialylation of E‐cadherin may participate in collective migration of uterine LE.ConclusionsThe study reveals a mechanism of uterine lumen closure by identifying that ST6GAL1‐mediated α2,6‐sialylation of cell adhesion molecules contributes to endometrial fold extension through regulating collective migration of uterine LE.

Cellular mechanisms and developmental compensation during tissue fusion in the secondary palate

Cleft lip with or without cleft palate (CL/P) is the most common congenital craniofacial anomaly with considerable morbidity in affected children. Though extensive studies have established the basic histological and genetic mechanisms of palatal development, the cellular mechanisms that regulate secondary palate development are still not completely understood. Palate fusion is the crucial final step of palate development, in which the medial edge epithelial cells (MEE) make contact to form a midline epithelial seam (MES), which is subsequently removed to allow mesenchymal continuity. Though much progress has been made on understanding the cellular mechanisms of secondary palate fusion, major questions remain in part due to limitations in imaging technologies. Here we utilized new processing and imaging methods to visualize dynamic cell behaviors underlying secondary palate fusion temporally and spatially. We apply these methods to the study of mouse mutants in which apoptosis, or actomyosin contractility have been conditionally disrupted in the secondary palate epithelium. Our results indicate that apoptosis and cell extrusion contribute to MES removal, but that cellular compensation can overcome their loss. Our studies also reveal a unique form of collective epithelial cell migration that is critical for proper completion of secondary palate fusion and suggesting that secondary palate fusion may be a valuable model for understanding how collective epithelial migration occurs in other developmental and disease contexts. Finally, our observations show that genetically ablating actomyosin contractility disrupts MES removal by disrupting collective epithelial cell migration. Overall, our studies suggest a model of compensatory cellular mechanisms in epithelium removal during palate fusion.

Collective epithelial cell migration in secondary palate fusion

Cleft lip with or without cleft palate (CL/P) is the most common congenital craniofacial anomaly with considerable morbidity in affected children. Though extensive studies have established the basic histological and genetic mechanisms of palatal development, the cellular mechanisms that regulate secondary palate development are still not completely understood. Palate fusion is the crucial final step of palate development, in which the medial edge epithelial cells (MEE) make contact to form a midline epithelial seam (MES), which is subsequently removed to allow mesenchymal continuity. Though much progress has been made on understanding the cellular mechanisms of secondary palate fusion, major questions remain in part due to limitations in imaging technologies. Here we utilized new processing and imaging methods to visualize dynamic cell behaviors underlying secondary palate fusion temporally and spatially. We apply these methods to the study of mouse mutants in which apoptosis, or actomyosin contractility have been conditionally disrupted in the secondary palate epithelium. Our results indicate that apoptosis and cell extrusion contribute to MES removal, but that cellular compensation can overcome their loss. Our studies also reveal a unique form of collective epithelial cell migration that is critical for proper completion of secondary palate fusion and suggesting that secondary palate fusion may be a valuable model for understanding how collective epithelial migration occurs in other developmental and disease contexts.

Integrins αvβ5 and αvβ6 Mediate IL-4-induced Collective Migration in Human Airway Epithelial Cells.

A positive link between persistent cellular motion and a defective tight junction barrier allows increased antigenic penetration and contact between ligand-receptor pairs, leading to exacerbated allergic airway inflammation and remodeling. Given that collective cell migration involves cell-cell and cell-extracellular matrix adhesions, and given that IL-4 induces epithelial barrier dysfunction and decreases cell-extracellular matrix adhesions, we hypothesized that IL-4 may induce collective migration in the well-differentiated primary human nasal epithelial cells (HNECs). Well-differentiated HNECs were treated with IL-4, and the effects of IL-4 on cell migration were investigated using genetic and pharmacological approaches, live-cell imaging, a vertex model, and immunostaining. IL-4 disrupted the expression and localization of the tight junction proteins zonula occludens 1 and occludin, and it induced the cleavage and asymmetric distribution of E-cadherin in the HNEC layers. It also induced collective epithelial migration and cell shape changes driven by actin cytoskeleton reorganization. In addition, the effect of IL-4 on collective HNEC migration was reversed by pharmacologic and genetic inhibition of the αv-integrin-activating enzyme furin, and function-blocking antibodies for αvβ5 or αvβ6. In IL-4-stimulated cells, both anti-αvβ5 and anti-αvβ6 inhibited the phosphorylation of focal adhesion kinase. Furthermore, both β5- and β6-integrins were enriched in basal cells in the injured airway epithelium with allergic rhinitis. These findings suggest that αvβ5 and αvβ6 serve as critical mechanoreceptors in IL-4-induced collective HNEC migration through the focal adhesion kinase signaling pathway. These results have implications for targeting treatment of exacerbation of respiratory allergic diseases.

Numerical investigation of the role of intercellular interactions on collective epithelial cell migration.

During collective cell migration, the intercellular forces will significantly affect the collective migratory behaviors. However, the measurement of mechanical stresses exerted at cell-cell junctions is very challenging. A recent experimental observation indicated that the intercellular adhesion sites within a migrating monolayer are subjected to both normal stress exerted perpendicular to cell-cell junction surface and shear stress exerted tangent to cell-cell junction surface. In this study, an interfacial interaction model was proposed to model the intercellular interactions for the first time. The intercellular interaction model-based study of collective epithelial migration revealed that the direction of cell migration velocity has better alignment with the orientation of local principal stress at higher maximum shear stress locations in an epithelial monolayer sheet. Parametric study of the effects of adhesion strength indicated that normal adhesion strength at the cell-cell junction surface has dominated effect on local alignment between the direction of cell velocity vector and the principal stress orientation, while the shear adhesion strength has little effect, which provides compelling evidence to help explain the force transmission via cell-cell junctions between adjacent cells in collective cell motion and provides new insights into "adhesive belt" effects at cell-cell junction.

Direction-dependent contraction forces on cell boundaries induce collective migration of epithelial cells within their sheet.

During early embryonic development, epithelial cells form a monolayer sheet and migrate in a definite direction. This phenomenon, called epithelial cell migration, is an important topic in developmental biology. A characteristic feature of this process is attachment to adjacent cells during migration, which is necessary for maintaining the integrity of the sheet. However, it is unclear how these cohesive cells migrate without breaking their attachments. A mechanism for this phenomenon was recently proposed, in which direction-dependent contraction forces acting on cell boundaries induce unidirectional epithelial migration. In this review, we examine this proposed mechanism from various aspects and provide theoretical background for the collective migration of epithelial cells. This information may be helpful for investigators to realize the basic principles underlying collective epithelial migration and devise new mechanisms for it.

β-Pix directs collective migration of anterior visceral endoderm cells in the early mouse embryo.

Collective epithelial migration is important throughout embryonic development. The underlying mechanisms are poorly understood but likely involve spatially localized activation of Rho GTPases. We previously reported that Rac1 is essential for generating the protrusive activity that drives the collective migration of anterior visceral endoderm (AVE) cells in the early mouse embryo. To identify potential regulators of Rac1, we first performed an RNAi screen of Rho family exchange factors (guanine nucleotide exchange factor [GEF]) in an in vitro collective epithelial migration assay and identified β-Pix. Genetic deletion of β-Pix in mice disrupts collective AVE migration, while high-resolution live imaging revealed that this is associated with randomly directed protrusive activity. We conclude that β-Pix controls the spatial localization of Rac1 activity to drive collective AVE migration at a critical stage in mouse development.

Collective epithelial migration drives kidney repair after acute injury.

Acute kidney injury (AKI) is a common and significant medical problem. Despite the kidney’s remarkable regenerative capacity, the mortality rate for the AKI patients is high. Thus, there remains a need to better understand the cellular mechanisms of nephron repair in order to develop new strategies that would enhance the intrinsic ability of kidney tissue to regenerate. Here, using a novel, laser ablation-based, zebrafish model of AKI, we show that collective migration of kidney epithelial cells is a primary early response to acute injury. We also show that cell proliferation is a late response of regenerating kidney epithelia that follows cell migration during kidney repair. We propose a computational model that predicts this temporal relationship and suggests that cell stretch is a mechanical link between migration and proliferation, and present experimental evidence in support of this hypothesis. Overall, this study advances our understanding of kidney repair mechanisms by highlighting a primary role for collective cell migration, laying a foundation for new approaches to treatment of AKI.

Twist1 induces dissemination by activating an epithelial motility program that requires E‐cadherin (LB230)

Cancer metastasis begins with dissemination of cells from an epithelial tumor. Each tumor contains many mutations and a heterogeneous cell population. As such, it has been difficult to isolate the molecular requirements for a single epithelial cell to disseminate. One posited mechanism for dissemination is activation of an epithelial to mesenchymal transition (EMT), in which repression of the cell adhesion gene E‐cadherin is considered the driving molecular event. We sought to test the sufficiency of single gene perturbations to induce dissemination in primary murine mammary epithelium. Deletion of E‐cadherin disrupted simple architecture and branching morphogenesis but rarely resulted in dissemination. In contrast, expression of the EMT transcription factor Twist1 induced rapid dissemination of cells into the extracellular matrix. Twist1 induced acute transcriptional changes in cell‐matrix adhesion genes but not in epithelial‐specific genes, including E‐cadherin. Surprisingly, knockdown of E‐cadherin inhibited Twist1‐induced single cell dissemination and promoted collective epithelial migration. We conclude that Twist1 activates an epithelial motility program without requiring a transition to mesenchymal cell fate. To date, it remains unclear why some Twist1+ cells disseminate while many remain in the epithelium. We next seek to test the sufficiency of Twist1 to induce dissemination in distinct mammary epithelial subpopulations. We anticipate that cell type‐specific differences may suggest which cells within a heterogeneous tumor have the highest metastatic potential.

Twist1 induces dissemination by activating an epithelial motility program that requires E‐cadherin (479.2)

Cancer metastasis begins with dissemination of cells from an epithelial tumor. Each tumor contains many mutations and a heterogeneous cell population. As such, it has been difficult to isolate the molecular requirements for a single epithelial cell to disseminate. One posited mechanism for dissemination is activation of an epithelial to mesenchymal transition (EMT), in which repression of the cell adhesion gene E‐cadherin is considered the driving molecular event. We sought to test the sufficiency of single gene perturbations to induce dissemination in primary murine mammary epithelium. Deletion of E‐cadherin disrupted simple architecture and branching morphogenesis but rarely resulted in dissemination. In contrast, expression of the EMT transcription factor Twist1 induced rapid dissemination of cells into the extracellular matrix. Twist1 induced acute transcriptional changes in cell‐matrix adhesion genes but not in epithelial‐specific genes, including E‐cadherin. Surprisingly, knockdown of E‐cadherin inhibited Twist1‐induced single cell dissemination and promoted collective epithelial migration. We conclude that Twist1 activates an epithelial motility program without requiring a transition to mesenchymal cell fate. To date, it remains unclear why some Twist1+ cells disseminate while many remain in the epithelium. We next seek to test the sufficiency of Twist1 to induce dissemination in distinct mammary epithelial subpopulations. We anticipate that cell type‐specific differences may suggest which cells within a heterogeneous tumor have the highest metastatic potential.

Mechanical Stretch and PI3K Signaling Link Cell Migration and Proliferation to Coordinate Epithelial Tubule Morphogenesis in the Zebrafish Pronephros

Organ development leads to the emergence of organ function, which in turn can impact developmental processes. Here we show that fluid flow-induced collective epithelial migration during kidney nephron morphogenesis induces cell stretch that in turn signals epithelial proliferation. Increased cell proliferation was dependent on PI3K signaling. Inhibiting epithelial proliferation by blocking PI3K or CDK4/Cyclin D1 activity arrested cell migration prematurely and caused a marked overstretching of the distal nephron tubule. Computational modeling of the involved cell processes predicted major morphological and kinetic outcomes observed experimentally under a variety of conditions. Overall, our findings suggest that kidney development is a recursive process where emerging organ function “feeds back” to the developmental program to influence fundamental cellular events such as cell migration and proliferation, thus defining final organ morphology.

Abstract 4923: Understanding cancer invasion by the systems biological analysis of collective cell migration in 3D tissue models

Abstract Collective migration is essential for morphogenesis and wound repair in healthy tissue as well as for tissue invasion in cancer. In such migration cells move in supracellular, cadherin mediated units opposed to isolated ameoboid or mesenchymal movements. Understanding collective migration in healthy epithelia is a prerequisite for dissecting and analyzing invasion in carcinoma. We have set up a novel, easily reproducible 3D in vitro wound model of epidermal regeneration. The model is based on commercial tissue cultures and can thus be reproducibly manufactured in larger quantities. We used our model for a systematic analysis of wound reepithelialization by measuring cytokines and mRNA expression. Using virtual microscopy we could quantitatively capture the cellular intra-tissue streams during reepithelialization. We integrated our data in a systems biological multi-scale model. Our results uncover a sequence of different types of collective epithelial migration which occur during wound healing in an orchestrated manner. In cancer invasion these mechanisms are hijacked by the tumor in an emergent malignant exploit of the intrinsically healthy processes of epithelial morphogenesis and repair. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4923. doi:1538-7445.AM2012-4923

Cellular strategies and molecular regulation of normal and neoplastic epithelial morphogenesis

Epithelia are one of the fundamental tissues of the human body and comprise an essential functional element of each of the major organs. We seek to resolve epithelial morphogenesis into a series of molecularly regulated changes in the discrete properties and behaviors of individual cells. In the mammary gland this challenge is particularly acute, as the epithelial and stromal compartments are both completely restructured during pubertal branching morphogenesis and each round of pregnancy, lactation and involution. We have developed a suite of epithelial isolation, 3D culture and 4D, parallel timelapse microscopic techniques to enable the study of the cellular basis of epithelial morphogenesis in real-time. We have demonstrated that mammary ductal morphogenesis proceeds by a novel form of collective epithelial migration and involves the transient formation of a highly proliferative, basement membrane degrading, highly motile, stratified epithelial architecture. During morphogenesis, we have shown that normal mammary ducts share many organizational features with mammary carcinomas. We have now combined our imaging approaches with labeled, genetic mosaic analysis to reveal the cell behavioral consequences of gene deletion on individual cells. Our main focus is to identify deletions sufficient to permit dissemination of normal cells into the ECM.

Abstract SY02-02: Novel mechanisms regulating breast cancer progression, metastasis, and response to therapy

Abstract In 1863, Virchow hypothesized that the cancer originated at sites of chronic tissue injury and that the ensuing inflammation they cause enhances cell proliferation. While it is now clear that proliferation of cells alone does not cause cancer, sustained cell proliferation in an environment rich in inflammatory cells, growth factors, activated stroma and DNA damage promoting agents, certainly potentiates and/or promotes neoplastic risk. In fact, in some cases, the trigger for neoplastic progression may even come from signals within the stromal microenvironment. The stromal microenvironment consists of several cell types (fibroblasts, macrophages, vascular components, and inflammatory cells of the innate and acquired immune response), as well as the extracellular matrix that they elaborate and all the molecules that are concentrated and immobilized on it. All of these components communicate with each other and with the neoplastic cells to contribute to the aberrant tumor organ. We used genetic and imaging techniques to study the interaction between inflammatory and epithelial cancer cells during tumor progression. We visualized the behavior of the cancer cells and leukocytes in living, anaesthetized mice using a spinning disk confocal microscope. We found that leukocytes observed at the tumor-stroma interface are very motile whereas those within the tumor are relatively immotile. The inflammatory switch occurred in parallel with the malignant conversion of the tumor cells. Transcriptional regulation of epithelial differentiation, cell-cell interaction and motility was a key intrinsic event in tumor progression. Tumor cell progression was regulated by GATA3, a master regulator for luminal differentiation, which was lost upon malignant progression, when the tumor cells disseminated throughout the body. Restoration of GATA3 into the malignant cells prevented the earliest stage of tumor metastasis, tumor cell dissemination, and altered the stromal response including decreasing angiogenesis. Zeppo1, a transcriptional repressor present in an amplicon on human chromosome 8p12, regulated epithelial cell adhesion and migration, enhancing later events in metastasis. The inflammatory cells modulate the tumor ecology, altering vascular permeability and responses to chemotherapy. The dynamic interplay of tumor cells and host cells responding to the tumor contribute to tumor evolution and evasion of therapeutic responses.Our data support the hypothesis that that both extrinsic and intrinsic mechanisms regulate tumor progression. Egeblad, M., A. J. Ewald, et al. (2008). Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy. Dis. Model. Mech. 1:155-167. PMID: 1904807 Ewald, A.J., A. Brenot, M. Duong, B.S. Chan & Z. Werb (2008). Collective epithelial migration and cell rearrangements drive mammary branching morphogenesis. Dev. Cell. 14:570-581. PMID: 18410732. Kouros-Mehr, H., S. K. Bechis, et al. (2008). GATA-3 links tumor differentiation and dissemination in a luminal breast cancer model. Cancer Cell. 13:141-52. PMID: 18242514. Lu, P. & Z. Werb (2008). Patterning mechanisms of branched organs. Science. 322:1506-1509. PMID: 19056977. Welm, B.E, G. J. P. Dijkgraaf, A. S. Bledau, A. L. Welm & Z. Werb (2008). Lentiviral transduction of stem cells for genetic analysis of mammary development and breast cancer. Cell Stem Cell. 2:90-102. PMID: 18371425. Citation Format: Zena Werb. Novel mechanisms regulating breast cancer progression, metastasis, and response to therapy [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY02-02

Molecular regulation of collective epithelial migration during mammary branching morphogenesis

Molecular regulation of collective epithelial migration during mammary branching morphogenesis