Cell Delamination Research Articles

Proximate larval epidermal cell layer generates forces for Pupal thorax closure in Drosophila.

During tissue closures, such as embryonic dorsal closure in Drosophila melanogaster, a proximate extra-embryonic layer, amnioserosa, generates forces that drive migration of the flanking lateral embryonic epidermis, thereby zip-shutting the embryo. Arguably, this paradigm of tissue closure is also recapitulated in mammalian wound healing wherein proximate fibroblasts transform into contractile myofibroblasts, develop cell junctions, and form a tissue layer de novo: contraction of the latter then aids in wound closure. Given this parallelism between disparate exemplars, we posit a general principle of tissue closure via proximate cell layer-generated forces. Here, we have tested this hypothesis in pupal thorax closure wherein 2 halves of the presumptive adult thorax of Drosophila, the contralateral heminotal epithelia, migrate over an underlying larval epidermal cell layer. We show that the proximate larval epidermal cell layer promotes thorax closure by its active contraction, orchestrated by its elaborate actomyosin network-driven epithelial cell dynamics, cell delamination, and death-the latter being prefigured by the activation of caspases. Larval epidermal cell dynamics generate contraction forces, which when relayed to the flanking heminota-via their mutual integrin-based adhesions-mediate thorax closure. Compromising any of these contraction force-generating mechanisms in the larval epidermal cell layer slows down heminotal migration, while loss of its relay to the flanking heminota abrogates the thorax closure altogether. Mathematical modeling further reconciles the biophysical underpinning of this emergent mechanism of thorax closure. Revealing mechanism of thorax closure apart, these findings show conservation of an essential principle of a proximate cell layer-driven tissue closure.

Draxin inhibits chick trunk neural crest delamination and migration by increasing cell adhesion.

The neural crest is a multipotent cell population that migrates extensively to play important roles during embryonic development. After acquiring motility, trunk neural crest cells delaminate from the spinal cord and migrate to various regions of the body. Several cellular adhesion molecules, such as vinculin, are involved in the regulation of neural crest delamination and migration. In the present study, we found that draxin could inhibit delamination and migration of neural crest cells from the chick spinal cord and abnormal aggregation of the migrating neural crest cells. In the presence of draxin, the resuspended neural crest regained its adhesive ability such that it was significantly increased. Overexpression of draxin caused increased vinculin expression in vivo. Our data indicate that draxin might control delamination and migration of chick trunk neural crest by increasing cell adhesion.

Apical contacts stemming from incomplete delamination guide progenitor cell allocation through a dragging mechanism.

The developmental strategies used by progenitor cells to allow a safe journey from their induction place towards the site of terminal differentiation are still poorly understood. Here, we uncovered a mechanism of progenitor cell allocation that stems from an incomplete process of epithelial delamination that allows progenitors to coordinate their movement with adjacent extra-embryonic tissues. Progenitors of the zebrafish laterality organ originate from the superficial epithelial enveloping layer by an apical constriction process of cell delamination. During this process, progenitors retain long-lasting apical contacts that enable the epithelial layer to pull a subset of progenitors on their way to the vegetal pole. The remaining delaminated cells follow the movement of apically attached progenitors by a protrusion-dependent cell-cell contact mechanism, avoiding sequestration by the adjacent endoderm, ensuring their collective fate and allocation at the site of differentiation. Thus, we reveal that incomplete delamination serves as a cellular platform for coordinated tissue movements during development.

High proliferation and delamination during skin epidermal stratification

The development of complex stratified epithelial barriers in mammals is initiated from single-layered epithelia. How stratification is initiated and fueled are still open questions. Previous studies on skin epidermal stratification suggested a central role for perpendicular/asymmetric cell division orientation of the basal keratinocyte progenitors. Here, we use centrosomes, that organize the mitotic spindle, to test whether cell division orientation and stratification are linked. Genetically ablating centrosomes from the developing epidermis leads to the activation of the p53-, 53BP1- and USP28-dependent mitotic surveillance pathway causing a thinner epidermis and hair follicle arrest. The centrosome/p53-double mutant keratinocyte progenitors significantly alter their division orientation in the later stages without majorly affecting epidermal differentiation. Together with time-lapse imaging and tissue growth dynamics measurements, the data suggest that the first and major phase of epidermal development is boosted by high proliferation rates in both basal and suprabasally-committed keratinocytes as well as cell delamination, whereas the second phase maybe uncoupled from the division orientation of the basal progenitors. The data provide insights for tissue homeostasis and hyperproliferative diseases that may recapitulate developmental programs.

146 A two-phase model of epidermal stratification: Lessons from centrosomes

The stratified skin epidermal barrier in mammals develops from a single-layered epithelium. The mechanisms initiating epidermal stratification and how the process is fueled are still not well-understood. Previous studies proposed a central role for perpendicular/asymmetric cell division orientation of the basal keratinocyte progenitors in epidermal stratification. Here, we used centrosomes, that organize the mitotic spindle, to test whether cell division orientation and stratification are linked. Genetic ablation of centrosomes from the developing epidermis led to the activation of the p53-dependent mitotic surveillance pathway resulting in reduced epidermal thickness and hair follicle arrest. Double mutant keratinocyte progenitors lacking both centrosomes and p53 rescued the epidermal phenotypes. Importantly, the mutant progenitors significantly altered their division orientation in the later developmental stages without affecting stratification or differentiation. Based on time-lapse imaging and analysis of tissue growth dynamics, our data suggested a two-phase model of epidermal stratification. The first and major phase of epidermal stratification is characterized by highly proliferating basal and suprabasal transit-amplifying cells as well as cell delamination, while the second phase may be uncoupled from the division orientation of the basal progenitors. The data provide insights for tissue homeostasis and hyperproliferative diseases that may recapitulate developmental programs.

Thermal-expansion offset for high-performance fuel cell cathodes.

One challenge for the commercial development of solid oxide fuel cells as efficient energy-conversion devices is thermo-mechanical instability. Large internal-strain gradients caused by the mismatch in thermal expansion behaviour between different fuel cell components are the main cause of this instability, which can lead to cell degradation, delamination or fracture1-4. Here we demonstrate an approach to realizing full thermo-mechanical compatibility between the cathode and other cell components by introducing a thermal-expansion offset. We use reactive sintering to combine a cobalt-based perovskite with high electrochemical activity and large thermal-expansion coefficient with a negative-thermal-expansion material, thus forming a composite electrode with a thermal-expansion behaviour that is well matched to that of the electrolyte. A new interphase is formed because of the limited reaction between the two materials in the composite during the calcination process, which also creates A-site deficiencies in the perovskite. As a result, the composite shows both high activity and excellent stability. The introduction of reactive negative-thermal-expansion components may provide a general strategy for the development of fully compatible and highly active electrodes for solid oxide fuel cells.

Fast Thermoresponsive Poly(oligoethylene glycol methacrylate) (POEGMA)-Based Nanostructured Hydrogels for Reversible Tuning of Cell Interactions.

Reactive electrospinning is demonstrated as a viable method to create fast-responsive and degradable macroporous thermoresponsive hydrogels based on poly(oligoethylene glycol methacrylate) (POEGMA). Hydrazide- and aldehyde-functionalized POEGMA precursor polymers were coelectrospun to create hydrazone cross-linked nanostructured hydrogels in a single processing step that avoids the need for porogens, phase separation-driving additives, or scaffold postprocessing. The resulting nanostructured hydrogels can respond reversibly and repeatedly to changes in external temperature within seconds, in contrast to the minutes-to-hours response time observed with bulk hydrogels. Furthermore, nearly quantitative cell delamination can be achieved within 2 min of incubation at 4 °C, resulting in the recovery of as many or more (as well as more proliferatively active) cells from the substrate relative to the conventional trypsinization protocol. The combined macroporosity, nanoscale feature size, and interfacial switching potential of these nanostructured hydrogels thus offer promise for manipulating cell-hydrogel interactions as well as other applications in which rapid responses to external stimuli are desirable.

Neuronal Delamination and Outer Radial Glia Generation in Neocortical Development.

During neocortical development, many neuronally differentiating cells (neurons and intermediate progenitor cells) are generated at the apical/ventricular surface by the division of neural progenitor cells (apical radial glial cells, aRGs). Neurogenic cell delamination, in which these neuronally differentiating cells retract their apical processes and depart from the apical surface, is the first step of their migration. Since the microenvironment established by the apical endfeet is crucial for maintaining neuroepithelial (NE)/aRGs, proper timing of the detachment of the apical endfeet is critical for the quantitative control of neurogenesis in cerebral development. During delamination, the microtubule–actin–AJ (adherens junction) configuration at the apical endfeet shows dynamic changes, concurrent with the constriction of the AJ ring at the apical endfeet and downregulation of cadherin expression. This process is mediated by transcriptional suppression of AJ-related molecules and multiple cascades to regulate cell adhesion and cytoskeletal architecture in a posttranscriptional manner. Recent advances have added molecules to the latter category: the interphase centrosome protein AKNA affects microtubule dynamics to destabilize the microtubule–actin–AJ complex, and the microtubule-associated protein Lzts1 inhibits microtubule assembly and activates actomyosin systems at the apical endfeet of differentiating cells. Moreover, Lzts1 induces the oblique division of aRGs, and loss of Lzts1 reduces the generation of outer radial glia (oRGs, also called basal radial glia, bRGs), another type of neural progenitor cell in the subventricular zone. These findings suggest that neurogenic cell delamination, and in some cases oRG generation, could be caused by a spectrum of interlinked mechanisms.

Deregulation of the Epithelial-to-Mesenchymal Transition Process Underlies Zic2-Linked Holoprosencephaly

Mutations in the Zic2 gene cause holoprosencephaly type 5, but the mechanisms that translate Zic2 mutations into this devastating pathology remain unclear. Here, we report that Zic2 is expressed in a few epiblast cells during gastrulation to become transiently upregulated in the primitive streak. Later, during neurulation, Zic2 is expressed in neural crest cells and downregulated as they delaminate from the neural tube. In combination with transcriptomic data from mutant embryos, chromatin occupancy profiles in gastrula and neural crest cells reveal that Zic2 regulates a large number of genes associated with the Wnt, cadherin and TGF-β pathways. Functional experiments in chick and mice demonstrate that Zic2 blocks the activation of the Wnt canonical pathway at the time that induces the TGF-β pathway to trigger cell delamination. All these experiments together demonstrate that Zic2 initiates epithelial-to-mesenchymal transition at early developmental stages.

Widely Used Mutants of eiger, Encoding the Drosophila Tumor Necrosis Factor, Carry Additional Mutations in the NimrodC1 Phagocytosis Receptor.

The process of apoptosis in epithelia involves activation of caspases, delamination of cells, and degradation of cellular components. Corpses and cellular debris are then rapidly cleared from the tissue by phagocytic blood cells. In studies of the Drosophila TNF, Eiger (Egr) and cell death in wing imaginal discs, the epithelial primordia of fly wings, we noticed that dying cells appeared to transiently accumulate in egr3 mutant wing discs, raising the possibility that their phagocytic engulfment by hemocytes was impaired. Further investigation revealed that lymph glands and circulating hemocytes from egr3 mutant larvae were completely devoid of NimC1 staining, a marker of phagocytic hemocytes. Genome sequencing uncovered mutations in the NimC1 coding region that are predicted to truncate the NimC1 protein before its transmembrane domain, and provide an explanation for the lack of NimC staining. The work that we report here demonstrates the presence of these NimC1 mutations in the widely used egr3 mutant, its sister allele, egr1, and its parental strain, Regg1GS9830. As the egr3 and egr1 alleles have been used in numerous studies of immunity and cell death, it may be advisable to re-evaluate their associated phenotypes.

π-SACS: pH Induced Self-Assembled Cell Sheets Without the Need for Modified Surfaces

The ability to form tissue-like constructs that have high cell density with proper cell-cell and cell-ECM interactions is critical for many applications including tissue models for drug discovery and tissue regeneration. Newly emerging bioprinting methods sometimes lack the high cellular density needed to provide biophysical cues to orchestrate cellular behavior to recreate tissue architecture and function. Alternate methods using self-assembly can be used to create tissue-like constructs with high cellular density and well-defined microstructure in the form of spheroids, organoids, or cell sheets. Cell sheets have a particularly interesting architecture in the context of tissue regeneration and repair as they can be applied as patches to integrate with surrounding tissues. Until now, the preparation of these sheets has involved culturing on specialized substrates that can be triggered by temperature or phase change (hydrophobic to hydrophilic) to release cells growing on them and form sheets. Here a new technique is proposed that allows delamination of cells and secreted ECM and rapid self-assembly into a cell sheet using a simple pH trigger and without the need to use responsive surfaces or applying external stimuli such as electrical and magnetic fields, only with routine tissue culture plates. This technique can be used with cells that are capable of syncytialization and fusion such as skeletal muscle cells and placenta cells. Using C2C12 myoblast cells we show that the pH trigger induces a rapid delamination of the cells as a continuous layer that self-assembles into a thick dense sheet. The delamination process has little effect on cell viability and maturation and preserves the ECM components that allow sheets to adhere to each other within a short incubation time enabling formation of thicker constructs when multiple sheets are stacked (double- and quadruple-layer constructs are formed here). These thick grafts can be used for regeneration purposes or as in vitro models.

Single layer low-temperature SOFC based on Ce0.8Sm0.2O2-δ- La0.25Sr0.75Ti1O3-δ-Ni0.8Co0.15Al0.05LiO2-δ composite material

This study reports a kind of single layer solid oxide fuel cell (SLSOFC) based on semiconductor-ionic conductor composite material which consists of Ce0.8Sm0.2O2-δ (SDC, ionic conductor), La0.25Sr0.75Ti1O3+δ (LST, n-type semiconductor) and Ni0.8Co0.15Al0.05LiO2-δ (NCAL, p-type semiconductor). It was found that the LST-SDC-NCAL composite based SLSOFC exhibited open circuit voltage (OCV) of more than 1 V and maximum power density of 222 mW cm−2 under 550 °C. In-situ Schottky junction in the device helps to prevent short circuiting as well as promote ion transport. Electrochemical impedance spectroscopy (EIS) analysis revealed that the ionic conductivity of the SLSOFC was about 0.09 S cm−1, and the corresponding activation energy is 0.7 eV. The cell performance was stable during 65 h without any significant degradation. Moreover, the SLSOFC possessed higher tolerance for temperature change than traditional three-layer SOFC due to the well match of thermal expansion coefficient between electrodes and electrolyte. This device is of great significance in preventing fuel cell delamination, simplifying manufacturing process and promoting its commercialization.

Investigation of Normal Cell and Cancer Cell Attachment and the Effects of Ganoderma Lucidum Using an Electric Impedance Sensing Technique

This research introduces an application of an electric impedance sensing technique to investigate cell attachment of normal epithelial cells (HaCAT) and cancerous cells (A431) before and after addition of Ganoderma Lucidum (reishi). In this study, an impedance sensing system is used to measure and characterize real-time changes in electric impedance (resistance and capacitance) with respect to an alternating current (AC) applied to HaCAT and A431 cell colonies. The impedance data is related to the properties of cell spreading, attachment, and delamination. The effect of reishi at dosages of 0.005, 0.01, and 0.02 mg/ml on these cellular properties was inferred from impedance data. The initial impedance data show that resistance is greater for A431 cells than HaCAT cells and that capacitance for A431 cells is less than the capacitance for HaCAT cells. Further, the data shows the resistance for HaCAT cells and for A431 cells increases with time, and the capacitance for both decreases with time. The impedance data analysis shows that reishi plays no role in altering the impedance of the cellular matrix. At most, reishi serves as a lubricant to allow partial detachment and reattachment of HaCAT cell-to-cell bonds, thus reordering (< entropy) the cellular matrix. This effect is not seen in A431 cell colonies.

RNA-Seq analysis on ets1 mutant embryos of Xenopus tropicalis identifies microseminoprotein beta gene 3 as an essential regulator of neural crest migration.

The proto-oncogene ets1 is highly expressed in the pre-migratory and migratory neural crest (NC), and has been implicated in the delamination and migration of the NC cells. To identify the downstream target genes of Ets1 in this process, we did RNA sequencing (RNA-Seq) on wild-type and ets1 mutant X. tropicalis embryos. A list of genes with significantly differential expression was obtained by analyzing the RNA-Seq data. We validated the RNA-Seq data by quantitative PCR, and examined the expression pattern of the genes identified from this assay with whole mount in situ hybridization. A majority of the identified genes showed expression in migrating NC. Among them, the expression of microseminoprotein beta gene 3 (msmb3) was positively regulated by Ets1 in both X. laevis and X. tropicalis. Knockdown of msmb3 with antisense morpholino oligonucleotides or disruption of msmb3 by CRISPR/Cas9 both impaired the migratory streams of NC. Our study identified msmb3 as an Ets1 target gene and uncovered its function in maintaining neural crest migration pattern.

ROS Regulate Caspase-Dependent Cell Delamination without Apoptosis in the Drosophila Pupal Notum.

SummaryThorax fusion occurs in the midline of the Drosophila pupal notum and involves epithelial cell delamination requiring apoptotic signaling. By genetic screening, we found that NADPH oxidases (Nox and Duox) associated with superoxide anion (O˙-2) are responsible for caspase-3 activation and delamination. We observed that Nox is upregulated in cells that undergo delamination and that delamination depends on caspase activation. However, the cell morphology and the almost complete lack of propidium iodide incorporation suggested little membrane disruption and signified apoptotic modulation. These results demonstrate that most delaminating cells undergo caspase activation, but this activation is not sufficient for apoptosis. We showed that the expression of Catalase, encoding an H2O2 scavenger in the cytosol, increases delamination and induces apoptotic nuclear fragmentation in caspase-3-activated cells. These findings suggest that the roles of O˙-2 and intracellular H2O2 for delamination differs before and after caspase-3 activation, which involves live cell delamination.

CAM-Delam: an in vivo approach to visualize and quantify the delamination and invasion capacity of human cancer cells

The development of metastases is the major cause of cancer related death. To develop a standardized method that define the ability of human cancer cells to degrade the basement membrane, e.g. the delamination capacity, is of importance to assess metastatic aggressiveness. We now present the in vivo CAM-Delam assay to visualize and quantify the ability of human cancer cells to delaminate and invade. The method includes seeding cancer cells on the chick chorioallantoic membrane (CAM), followed by the evaluation of cancer-induced delamination and potential invasion within hours to a few days. By testing a range of human cancer cell lines in the CAM-Delam assay, our results show that the delamination capacity can be divided into four categories and used to quantify metastatic aggressiveness. Our results emphasize the usefulness of this assay for quantifying delamination capacity as a measurement of metastatic aggressiveness, and in unraveling the molecular mechanisms that regulate delamination, invasion, formation of micro-metastases and modulations of the tumor microenvironment. This method will be useful in both the preclinical and clinical characterization of tumor biopsies, and in the validation of compounds that may improve survival in metastatic cancer.

Fascin regulates protrusions and delamination to mediate invasive, collective cell migration in vivo.

The actin bundling protein Fascin is essential for developmental cell migrations and promotes cancer metastasis. In addition to bundling actin, Fascin has several actin-independent roles; how these other functions contribute to cell migration remains unclear. Border cell migration during Drosophila oogenesis provides an excellent model to study Fascin's various roles during invasive, collective cell migration. On-time border cell migration during Stage 9 requires Fascin (Drosophila Singed). Fascin functions not only within the migrating border cells, but also within the nurse cells, the substrate for this migration. Fascin genetically interacts with the actin elongation factor Enabled to promote on-time Stage 9 migration and overexpression of Enabled suppresses the defects seen with loss of Fascin. Loss of Fascin results in increased, shorter and mislocalized protrusions during migration. Additionally, loss of Fascin inhibits border cell delamination and increases E-Cadherin (Drosophila Shotgun) adhesions on both the border cells and nurse cells. Overall, Fascin promotes on-time border cell migration during Stage 9 and contributes to multiple aspects of this invasive, collective cell migration, including both protrusion dynamics and delamination. These findings have implications beyond Drosophila, as border cell migration has emerged as a model to study mechanisms mediating cancer metastasis.

MMP14 is required for delamination of chick neural crest cells independently of its catalytic activity.

Matrix metalloproteinases have a broad spectrum of substrates ranging from extracellular matrix components and adhesion molecules to chemokines and growth factors. Despite being mostly secreted, MMPs have been detected in the cytosol, the mitochondria or the nucleus. Although most of the attention is focused on their role in matrix remodeling, the diversity of their substrates and their complex trafficking open the possibility for non-canonical functions. Yet in vivo examples and experimental demonstration of the physiological relevance of such activities are rare. Here, we have used chick neural crest (NC) cells, a highly migratory stem cell population likened to invasive cancer cells, as a model for physiological epithelial-mesenchymal transition (EMT). We demonstrate that MMP14 is required for NC delamination. Interestingly, this role is independent of its cytoplasmic tail and of its catalytic activity. Our in vivo data indicate that, in addition to being a late pro-invasive factor, MMP14 is also likely to be an early player, owing to its role in EMT.

Regulation of cranial neural crest cell fate by Irf6 and Twist1 interaction

A wide variety of common craniofacial birth defects and pediatric cancers arise from errors in the development of cranial neural crest cells (CNCC) that give rise to craniofacial skeletal and peripheral nervous systems. Identification of the genetic and environmental factors that regulate cell fate of CNCCs is crucial for uncovering the causes behind craniofacial disorders. Our lab has identified a dual‐transcription factor pathway that regulates CNCCs during EMT and migration. Initially, we identified a causative mutation in a TWIST1 binding site within an Interferon Regulatory Factor 6 (IRF6) enhancer in a multi‐generational family affected with syndromic cleft lip and palate called Van der Woude syndrome. Our subsequent animal work has shown that Twist1 and Irf6 genetically interact during early craniofacial development, and that the compound heterozygotes exhibit craniofacial abnormalities. We recently demonstrated that Irf6 is required for neural tube and CNCC development and that it interacts with Twist1 during EMT and migration of CNCCs. We also showed that TWIST1 is highly phosphorylated in CNCCs and its phosphorylation is crucial for regulating Irf6 and potentially miR10 family members in CNCC‐derived craniofacial tissues. Our recent in‐vivo and neural tube explants using dual fluorescent cell tracing system showed that Twist1 conditional knockout (CKO) in CNCCs significantly reduced cell delamination and disrupted EMT. The detached Twist1 CKO in CNCCs retained their epithelial signatures and migrated as clusters of epithelial‐like cells over significantly shorter distances. Finally, Twist1 phospho‐incompetent mice for two serine residues were recently generated to determine their impact on TWIST1 activity. Our preliminary results showed that Twist1S68A/S68A phospho‐mutant mice have craniofacial bone defects and forebrain hemorrhage.Support or Funding InformationUTHealth start‐up funds and a small grant from theRolanette and Berdon Lawrence Bone Disease Program of Texas

Crumbs2 mediates ventricular layer remodelling to form thespinal cord central canal.

In the spinal cord, the central canal forms through a poorly understood process termed dorsal collapse that involves attrition and remodelling of pseudostratified ventricular layer (VL) cells. Here, we use mouse and chick models to show that dorsal ventricular layer (dVL) cells adjacent to dorsal midline Nestin(+) radial glia (dmNes+RG) down-regulate apical polarity proteins, including Crumbs2 (CRB2) and delaminate in a stepwise manner; live imaging shows that as one cell delaminates, the next cell ratchets up, the dmNes+RG endfoot ratchets down, and the process repeats. We show that dmNes+RG secrete a factor that promotes loss of cell polarity and delamination. This activity is mimicked by a secreted variant of Crumbs2 (CRB2S) which is specifically expressed by dmNes+RG. In cultured MDCK cells, CRB2S associates with apical membranes and decreases cell cohesion. Analysis of Crb2F/F/Nestin-Cre+/- mice, and targeted reduction of Crb2/CRB2S in slice cultures reveal essential roles for transmembrane CRB2 (CRB2TM) and CRB2S on VL cells and dmNes+RG, respectively. We propose a model in which a CRB2S-CRB2TM interaction promotes the progressive attrition of the dVL without loss of overall VL integrity. This novel mechanism may operate more widely to promote orderly progenitor delamination.